Mhc class i epitope delivering polypeptides and cell-targeted molecules for direct cell killing and immune stimulation via mhc class i presentation and methods regarding the same

ABSTRACT

The present invention is directed to T-cell epitope delivering polypeptides which deliver one or more CD8+ T-cell epitopes to the MHC class I presentation pathway of a cell, including toxin-derived polypeptides which comprise embedded T-cell epitopes and are de-immunized. The present invention provides cell-targeted, CD8+ T-cell epitope delivering molecules for the targeted delivery of cytotoxicity to certain cells, e.g., infected or malignant cells, for the targeted killing of specific cell types, and the treatment of a variety of diseases, disorders, and conditions, including cancers, immune disorders, and microbial infections. The present invention also provides methods of generating polypeptides capable of delivering one or more heterologous T-cell epitopes to the MHC class I presentation pathway, including polypeptides which are 1) B-cell and/or CD4+ T-cell de-immunized, 2) comprise embedded T-cell epitopes, and/or 3) comprises toxin effectors which retain toxin functions.

FIELD OF THE INVENTION

The present invention relates generally to methods of modifyingpolypeptides to introduce the ability of the polypeptide to deliver aheterologous T-cell epitope for MHC class I presentation by a chordatecell and the polypeptides made using these methods. More specifically,the invention relates to methods of modifying polypeptides comprisingproteasome delivery effector functions into heterologous, T-cell epitopedelivering polypeptides that differ in their immunogenic properties fromtheir parent molecules by the addition of one or more T-cellepitope-peptides which can be recognized by a MHC class I molecule andbe presented on a cell surface by the MHC class I system of a chordatecell. Certain methods of the present invention relate to methods ofmodifying polypeptides to reduce antigenicity and/or immunogenicity viathe introduction of one or more T-cell epitopes. In another aspect, thepresent invention relates to polypeptides created using methods of theinvention and cell-targeted molecules comprising polypeptides createdusing methods of the invention. The cell-targeted molecules of thepresent invention may be used for numerous applications such as, e.g.,the diagnosis and treatment of a variety of diseases, disorders, andconditions, such as, e.g., cancers, tumors, other growth abnormalities,immune disorders, and microbial infections.

BACKGROUND

The immune systems of chordates, such as amphibians, birds, fish,mammals, reptiles, and sharks, constantly scan both the extracellularand intracellular environments for exogenous molecules in an attempt toidentify the presence of particularly threatening foreign molecules,cells, and pathogens. The Major Histo-Compatibility (MHC) systemfunctions in chordates as part of the adaptive immune system (Janeway'sImmunobiology (Murphy K, ed., Garland Science, 8^(th) ed., 2011)).Within a chordate, extracellular antigens are presented by the MHC classII system, whereas intracellular antigens can be presented by the MHCclass I system.

Generally, the administration of exogenous peptides, polypeptides, orproteins to a cell results in these molecules not entering the cell dueto the physical barrier of the plasma membrane. In addition, thesemolecules are often degraded into smaller molecules by extracellularenzymatic activities on the surfaces of cells and/or in theextracellular milieu. Polypeptides and proteins that are internalizedfrom the extracellular environment by endocytosis are commonly degradedby lysosomal proteolysis as part of an endocytotic pathway involvingearly endosomes, late endosomes, and lysosomes. Polypeptides andproteins that are internalized from the extracellular environment byphagocytosis are commonly degraded by a similar pathway ending withphagolysosomes

The MHC class II pathway presents antigenic peptides derived frommolecules in the extracellular space, commonly after phagocytosis andprocessing by specialized antigen presenting cells; these cells can beprofessional antigen presenting cells or other antigen presenting cells,such as, e.g., dendritic cells (DCs), mononuclear phagocytes (MNPCs),certain endothelial cells, and B-lymphocytes (B-cells). These antigenpresenting cells display certain peptides complexed with MHC class IImolecules on their cell surface for recognition by CD4 positive (CD4+)T-lymphocytes (T-cells). On the other hand, the MHC class I systemfunctions in most cells in a chordate to present antigenic peptides froman intracellular space, commonly the cytosol, for recognition by CD8+T-cells.

The MHC class I system plays an essential role in the immune system byproviding antigen presentation of intracellular antigens (Cellular andMolecular Immunology (Abbas A, ed., Saunders, 8^(th) ed., 2014)). Thisprocess is thought to be an important part of the adaptive immune systemwhich evolved in chordates primarily to protect against neoplastic cellsand microbial infections involving intracellular pathogens; however,certain damaged cells can be removed by this process as well. Thepresentation of an antigenic peptide complexed with a MHC class Imolecule sensitizes the presenting cells to targeted killing bycytotoxic T-cells (CTLs) via lysis, induced apoptosis, and/or necrosis.The presentation of specific peptide epitopes complexed with MHC class Imolecules plays a major role in stimulating and maintaining immuneresponses to cancers, tumors, and intracellular pathogens.

The MHC class I system continually functions to process and display onthe cell surface various intracellular epitopes, both self or non-self(foreign) and both peptide or lipid antigens. The MHC class I display offoreign antigens from intracellular pathogens or transformed cellssignal to CD8+ effector T-cells to mount protective T-cell immuneresponses. In addition, the MHC class I system continually functions topresent self peptide epitopes in order to establish and maintainimmunological tolerance.

Peptide epitope presentation by the MHC class I system involves fivemain steps: 1) generation of cytoplasmic peptides, 2) transport ofpeptides to the lumen of the endoplasmic reticulum (ER), 3) stablecomplex formation of MHC class I molecules bound to certain peptides, 4)display of those stable peptide-MHC class I molecule complexes(peptide-MHC class I complexes) on the cell surface, and 5) recognitionof certain antigenic, presented peptide-MHC class I complexes byspecific CD8+ T-cells, including specific CTLs.

The recognition of a presented antigen-MHC class I complex by a CD8+T-cell leads to CD8+ T-cell activation, clonal expansion, anddifferentiation into CD8+ effector cells, including CTLs which targetfor destruction cells presenting specific epitope-MHC class I complexes.This leads to the creation of a population of specific CD8+ effectorcells, some of which can travel throughout the body to seek and destroycells displaying a specific epitope-MHC class I complex.

The MHC class I system is initiated with a cytosolic peptide. Theexistence of peptides in the cytosol can occur in multiple ways. Ingeneral, peptides presented by MHC class I molecules are derived fromthe proteasomal degradation of intracellular proteins and polypeptides.The MHC class I pathway can begin with transporters associated withantigen processing proteins (TAPs) associated with the ER membrane. TAPstranslocate peptides from the cytosol to the lumen of the ER, where theycan then associate with empty MHC class I molecules. TAPs translocatepeptides which most commonly are of sizes around 8-12 amino acidresidues but also including 6-40 amino acid residues (Koopmann J et al.,Eur J Immunol 26: 1720-8 (1996)).

The MHC class I pathway can also be initiated in the lumen of the ER bya pathway involving transport of a protein, polypeptide, or peptide intothe cytosol for processing and then re-entry back into the ER viaTAP-mediated translocation.

The peptides transported from the cytosol into the lumen of the ER byTAP are then available to be bound by different MHC class I molecules.In the lumen of the ER, a multi-component peptide loading machine, whichinvolves TAPs, helps assemble stable peptide-MHC class I moleculecomplexes and further process peptides in some instances, especially bycleavage into optimal sized peptides in a process called trimming (seeMayerhofer P, Tampé R, J Mol Biol pii S0022-2835 (2014)). In the ER,different MHC class I molecules tightly bind using highly specificimmunoglobulin-type, antigen-binding domains to only those specificpeptides for which the MHC class I molecule has a stronger affinity.Then the peptide-MHC class I complex is transported via the secretorypathway to the plasma membrane for presentation to the extracellularenvironment and recognition by CD8+ T-cells.

Recognition by a CD8+ T-cell of an epitope-MHC class I complex initiatesprotective immune responses which ultimately ends in the death of thepresenting cell due to the cytotoxic activity of one or more CTLs. CTLsexpress different T-cell receptors (TCRs) with differing specificities.The MHC alleles are highly variable, and the diversity conferred bythese polymorphisms can influence recognition by T-cells in two ways: byaffecting the binding of peptide antigens and by affecting the contactregions between the MHC molecule and TCRs. In response to antigen-MHCclass I molecule complex recognition by a CTL via its particular cellsurface TCR, the CTL will kill the antigen-MHC class I complexpresenting cell primarily via cytolytic activities mediated by thedelivery of perforin and/or granzyme into the presenting cell. Inaddition, the CTL will release immuno-stimulatory cytokines, such as,e.g., interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF),macrophage inflammatory protein-1 beta (MIP-1beta), and interleukinssuch as IL-17, IL-4, and IL-22. Furthermore, activated CTLs canindiscriminately kill proximal to epitope-MHC class I complex presentingcell which activated them regardless of the proximal cell's presentpeptide-MHC class I complex repertoire (Wiedemann A et al., Proc NatlAcad Sci USA 103: 10985-90 (2006)). These epitope-MHC class I complexinduced immune responses could conceivably be harnessed by therapeuticsto kill certain cell-types within a patient as well as sensitize theimmune system to other proximal cells.

The MHC class I presentation pathway could be exploited by varioustherapeutics in order to induce desired immune responses; however, thereare several barriers to developing such a technology, including, e.g.,delivery through the cell plasma membrane; escaping the endocytoticpathway and destruction in the lysosome; and generally avoiding thesequestration, modification, and/or destruction of foreign polypeptidesby the targeted cell (Sahay G et al., J Control Release 145: 182-195(2010); Fuchs H et al., Antibodies 2: 209-35 (2013)).

In addition, the effectiveness of polypeptide-comprising therapeutics,e.g. polypeptide based biologics and biopharmaceuticals, is oftencurtailed by undesirable immune responses generated in recipients inresponse to the therapeutics. Virtually all polypeptide-basedtherapeutics induce some level of immune response after administrationto a mammalian subject. Different levels of immune responses include theproduction of low-level, low-affinity and transient immunoglobulin-Mantibodies to high-level, high-affinity immunoglobulin-G antibodies. Theimmunogenicity of a therapeutic might cause unwanted immune responses inrecipients which reduce therapeutic efficacy, adversely alterpharmacokinetics, and/or result in hypersensitivity reactions,anaphylaxis, anaphylactoid reactions, or infusion reactions among otherconsequences (see Buttel I et al., Biologicals 39: 100-9 (2011)).

For example, a polypeptide-based therapeutic can cause a recipient tocreate antibodies against antigenic sites in the therapeutic (sometimescalled neutralizing antibodies or anti-drug antibodies). Immuneresponses generating antibodies recognizing a therapeutic can resultimmunological resistance to the effect(s) of the therapeutic. Inaddition, cross-reactions between anti-therapeutic antibodies withendogenous factors can result in undesirable clinical outcomes.

Polypeptide-based therapeutics with polypeptide sequences derived fromspecies distantly related to the recipient, such as when the recipientis a mammal and the polypeptide sequences are derived from a plant ormicroorganism, tend to be aggressively targeted by the recipient'simmune system (see, Sauerborn M et al., Trends Pharmacol Sci 31: 53-9(2010), for review). Vertebrate immune systems have adapted to recognizeforeign polypeptide sequences with both innate and adaptive immunesystems. Thud, the administration of a polypeptide to a vertebrate fromthe same species of vertebrate can be recognized as non-self and elicitan immune response, such as, e.g., administering to a human apolypeptide comprising a recombinant junction of two heterologous humanpolypeptide sequences.

Therefore, when designing polypeptide-containing therapeutics it isoften desirable to attempt to minimize the immunogenicity of thetherapeutic to prevent and/or reduce the occurrence of undesired immuneresponses in subjects undergoing therapeutic treatment. In particular,polypeptide regions in therapeutics likely to produce B-cell and/orT-cell antigenicity and/or immunogenicity are targeted for removal,suppression, and minimization.

Both B-cell and T-cell epitopes can be predicted in a given polypeptidesequence in silico using software (see, Bryson C et al., BioDrugs 24:1-8 (2010), for review). For example, software called EpiMatrix (EpiVax,Inc., Providence, R.I., U.S.) was successfully used to predict T-cellimmunogenicity in recombinant proteins (De Groot A et al., Dev Biol(Basel) 122: 171-94 (2005); Koren E et al., Clin Immunol 124: 26-32(2007)).

Many approaches, such as the elimination of antigenic and/or immunogenicepitopes by truncation or mutation, have been described for reducing theimmunogenicity of polypeptide-containing therapeutics (Tangri S et al.,J Immunol 174: 3187-96 (2005); Mazor R et al., Proc Natl Acad Sci USA109: E3597-603 (2012); Yumura K et al., Protein Sci 22: 213-21 (2012)).Foreign polypeptides can be recognized with exquisite specificity by theadaptive immune system via immune epitopes often present at a smallnumber of discrete sites on the surface of the polypeptide. However,antibody-binding affinity can be dominated by interactions with a smallnumber of specific amino acids within an epitope. Thus, modifications ofthe crucial amino acids in a polypeptide which disrupt an immunogenicepitope can reduce immunogenicity (Laroche Y et al., Blood 96: 1425-32(2000)). Modifications which disrupt epitope recognition include aminoacid deletions, substitutions, and epitope masking with non-immunogenicconjugates.

For the development of polypeptide-based therapeutics, it is desirableto avoid inducing B-cell mediated immune responses and the production ofneutralizing antibodies in patients because it reduces the effectivenessof the therapy, changes the dose-effect profile, and limits the numberof doses a patient can receive (see Lui W et al., Proc Natl Acad Sci USA109: 11782-7 (2012)).

Thus, it would be desirable to have methods of creating novel T-cellepitope delivering polypeptides which can deliver one or more T-cellepitopes to the MHC class I presentation pathway of a cell. It wouldalso be desirable to have polypeptides which under physiologicalconditions can deliver a T-cell epitope to the interior of a target cellto initiate desirable T-cell mediated immune responses but do not induceundesirable immune responses while in extracellular spaces, such as,e.g., the creation of inhibitory antibodies. Thus, it would be desirableto have T-cell epitope delivering polypeptides in which one or more CD8+T-cell epitopes are added and one or more B-cell and/or CD4+ T-cellepitopes are abolished.

It would also be desirable to have cell-targeted, CD8+ T-cell epitopedelivering molecules for the targeted delivery of cytotoxicity tospecific cell types, e.g., infected or malignant cells. In addition, itwould be desirable to have cell-targeted, CD8+ T-cell epitope deliveringmolecules which exhibit reduced B-cell immunogenicity. Once the T-cellimmunogenic peptide(s) delivered by the cell-targeted molecule arepresented to the surface of a target cell, the T-cell epitope can signalfor the destruction of the presenting cell by activating the recipient'sown immune system to recruit CD8+ T-cells. In addition, CD8+ T-cellsactivated by the target cell's displayed T-cell epitope-MHC class Icomplex can stimulate a wider immune response and alter themicro-environment (e.g. by release cytokines in a tumor or infectedtissue locus), such that other immune cells (e.g. effector T-cells) maybe recruited to the local area.

In addition, it would be desirable to have methods of creating novelT-cell epitope delivering polypeptides which are derived from toxins yetpreserving certain biological effector functions of the parental toxinpolypeptide, such as promoting cellular internalization, directingsubcellular routing, and/or toxin enzymatic activity. In addition, it isdesirable to have methods of engineering toxin-derived polypeptides byreplacing a B-cell epitope with a T-cell epitope as a means to bothreduce the likelihood of the polypeptide producing an undesirable immuneresponse and to increase the likelihood of inducing a desirable T-cellresponse directed to those targeted cells that internalize the toxinpolypeptide comprising molecule.

SUMMARY OF THE INVENTION

The present invention provides various embodiments of T-cell epitopedelivering polypeptides (referred to herein as “CD8+ T-cellhyper-immunized”) which as components of certain cell-targeted moleculeshave the ability to deliver a T-cell epitope for presentation by anucleated, target cell within a chordate. The present invention alsoprovides various embodiments of de-immunized, CD8+ T-cellhyper-immunized polypeptides which have reduced antigenic and/orimmunogenic potential in mammals regarding a B-cell and/or CD4+ T-cellepitope (referred to herein as “B-cell and/or CD4+ T-cellde-immunized”). The present invention also provides various embodimentsof cell-targeted, CD8+ T-cell epitope delivering molecules for thetargeted delivery of cytotoxicity to specific cell types, e.g., infectedor malignant cells within a chordate.

In addition, the present invention provides embodiments of methods ofgenerating novel polypeptides capable of delivering one or moreheterologous T-cell epitopes to the MHC class I presentation pathway ofa cell. The present invention also provides various embodiments ofmethods of generating variants of polypeptides by simultaneouslyreducing the probability of B-cell and/or CD4+ T-cell immunogenicitywhile increasing the probability of CD8+ T-cell immunogenicity. Thepresent invention also provides certain embodiments of the methods ofgenerating novel polypeptides capable of delivering one or moreheterologous T-cell epitopes to the MHC class I presentation pathway ofa cell, wherein the starting polypeptide comprises a toxin effectorregion and certain polypeptides produced by using the methods of theinvention result in polypeptides which retain toxin effector functions,such as, e.g., enzymatic activity and cytotoxicity.

The polypeptides of the present invention may be either CD8+ T-cellhyper-immunized or de-immunized or both. The de-immunized polypeptidesof the present invention may be either B-cell epitope de-immunized orT-cell de-immunized or both. The T-cell de-immunized polypeptides of thepresent invention may be either CD4+ T-cell de-immunized or CD8+ T-cellde-immunized or both. Certain embodiments of the polypeptides of thepresent invention comprise one or more heterologous T-cell epitopes. Incertain further embodiments of the polypeptides of the presentinvention, the one or more heterologous T-cell epitopes are CD8+ T-cellepitopes.

In certain embodiments, a polypeptide of the present invention comprisesan embedded or inserted heterologous T-cell epitope, wherein thepolypeptide is capable of intracellular delivery of the T-cell epitopefrom an early endosomal compartment to a proteasome of a cell in whichthe polypeptide is present. In certain further embodiments, thepolypeptide of the present invention further comprises a toxin-derivedpolypeptide capable of routing to a subcellular compartment of a cell inwhich the toxin-derived polypeptide is present selected from the groupconsisting of: cytosol, endoplasmic reticulum, and lysosome. In certainfurther embodiments, the polypeptide of the present invention comprisesa heterologous T-cell epitope is embedded or inserted in a toxin-derivedpolypeptide.

In certain embodiments, a polypeptide of the present invention comprisesa toxin-derived polypeptide comprising a toxin effector polypeptidecapable of exhibiting one or more toxin effector functions. In certainfurther embodiments, the toxin effector polypeptide is derived from atoxin selected from the group consisting of: ABx toxin, ribosomeinactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labileenterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein,pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin,sarcin, Shiga toxin, and subtilase cytotoxin.

In certain embodiments, the polypeptide of the present inventioncomprises the toxin effector polypeptide derived from amino acids 75 to251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, or amino acids 2 to389 of SEQ ID NO:45. In certain further embodiments, the polypeptide ofthe present invention comprises the Shiga toxin effector polypeptidederived from amino acids 1 to 241 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3. In certain further embodiments, the Shiga toxin effectorpolypeptide is derived from amino acids 1 to 251 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO:3. In certain further embodiments, the Shiga toxineffector polypeptide is derived from amino acids 1 to 261 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3.

In certain embodiments, a polypeptide of the present invention comprisesan embedded or inserted heterologous CD8+ T-cell epitope, wherein thepolypeptide is capable of intracellular delivery of the T-cell epitopeto a MHC class I molecule from an early endosomal compartment of a cellin which the polypeptide is present. In certain further embodiments, thepolypeptide further comprises a toxin-derived polypeptide capable ofrouting to a subcellular compartment of a cell in which the polypeptideis present selected from the group consisting of: cytosol, endoplasmicreticulum, and lysosome. In certain further embodiments, the polypeptideof the present invention comprises the heterologous CD8+ T-cell epitopein the toxin-derived polypeptide. In certain further embodiments, thepolypeptide of the present invention comprises the toxin-derivedpolypeptide comprising a toxin effector polypeptide capable ofexhibiting one or more toxin effector functions. In certain furtherembodiments, the polypeptide of the present invention comprises thetoxin effector polypeptide derived from a toxin selected from the groupconsisting of: ABx toxin, ribosome inactivating protein toxin, abrin,anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin,diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin,pertussis toxin, pokeweed antiviral protein, pulchellin, Pseudomonasexotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, andsubtilase cytotoxin. In certain embodiments, the polypeptide of thepresent invention comprises the toxin effector polypeptide derived fromamino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, oramino acids 2 to 389 of SEQ ID NO:45. In certain further embodiments,the polypeptide of the present invention comprises the Shiga toxineffector polypeptide derived from amino acids 1 to 241 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments, the Shigatoxin effector polypeptide is derived from amino acids 1 to 251 of SEQID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,the Shiga toxin effector polypeptide is derived from amino acids 1 to261 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

In certain embodiments, a polypeptide of the present invention comprisesa heterologous CD8+ T-cell epitope, wherein the polypeptide is capableof intracellular delivery of the T-cell epitope for presentation by aMHC class I molecule on the surface of a cell in which the polypeptideis present. In certain further embodiments, the polypeptide of thepresent invention comprises a toxin-derived polypeptide capable ofrouting to a subcellular compartment of a cell in which thetoxin-derived polypeptide is present selected from the group consistingof: cytosol, endoplasmic reticulum, and lysosome. In certain furtherembodiments, the polypeptide of the present invention comprises theheterologous CD8+ T-cell epitope in the toxin-derived polypeptide. Incertain further embodiments, the polypeptide of the present inventioncomprises the toxin-derived polypeptide comprising a toxin effectorpolypeptide capable of exhibiting one or more toxin effector functions.In certain further embodiments, the polypeptide of the present inventioncomprises the toxin effector polypeptide derived from a toxin selectedfrom the group consisting of: ABx toxin, ribosome inactivating proteintoxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin,claudin, diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin,pertussis toxin, pokeweed antiviral protein, pulchellin, Pseudomonasexotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, andsubtilase cytotoxin. In certain embodiments, the polypeptide of thepresent invention comprises the toxin effector polypeptide derived fromamino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, oramino acids 2 to 389 of SEQ ID NO:45. In certain further embodiments,the polypeptide of the present invention comprises the Shiga toxineffector polypeptide derived from amino acids 1 to 241 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments, the Shigatoxin effector polypeptide is derived from amino acids 1 to 251 of SEQID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,the Shiga toxin effector polypeptide is derived from amino acids 1 to261 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

In certain embodiments, a polypeptide of the present invention comprisesa proteasome delivering effector polypeptide associated with aheterologous CD8+ T-cell epitope, and capable of intracellular deliveryof the T-cell epitope for presentation by a MHC class I molecule on thesurface of a cell in which the polypeptide is present. In certainfurther embodiments, the polypeptide of the present invention comprisesa Shiga toxin effector polypeptide, wherein the heterologous CD8+ T-cellepitope is not fused directly to the amino-terminus of the Shiga toxineffector polypeptide. In certain further embodiments, the polypeptide ofthe present invention further comprises a second T-cell epitope embeddedor inserted into a B-cell epitope. In certain further embodiments, thepolypeptide of the present invention further comprises a toxin-derivedpolypeptide. In certain further embodiments, the polypeptide of thepresent invention further comprises the toxin-derived polypeptidecomprising a toxin effector polypeptide comprising the proteasomedelivering effector polypeptide and the second T-cell epitope. Incertain further embodiments, a polypeptide of the present inventioncomprises the toxin-derived polypeptide comprising a toxin effectorpolypeptide capable of exhibiting one or more toxin effector functions.In certain further embodiments, the polypeptide of the present inventioncomprises the toxin effector polypeptide derived from a toxin selectedfrom the group consisting of: ABx toxin, ribosome inactivating proteintoxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin,claudin, diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin,pertussis toxin, pokeweed antiviral protein, pulchellin, Pseudomonasexotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, andsubtilase cytotoxin. In certain embodiments, the polypeptide of 15 thepresent invention comprises the toxin effector polypeptide derived fromamino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3, oramino acids 2 to 389 of SEQ ID NO:45. In certain further embodiments,the polypeptide of the present invention comprises the Shiga toxineffector polypeptide derived from amino acids 1 to 241 of SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments, the Shigatoxin effector polypeptide is derived from amino acids 1 to 251 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In certain further embodiments,the Shiga toxin effector polypeptide is derived from amino acids 1 to261 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.

In certain embodiments of the methods of the present invention is amethod of increasing CD8+ T-cell immunogenicity of a polypeptide capableof intracellular routing to a subcellular compartment of a cell in whichthe polypeptide is present selected from the group consisting of:cytosol, endoplasmic reticulum, and lysosome; the method comprising thestep of: embedding or inserting a heterologous CD8+ T-cell epitope inthe polypeptide. In certain further embodiments, the method comprisesthe embedding or inserting step wherein the embedding or inserting in anendogenous B-cell epitope, an endogenous CD4+ T-cell epitope, and/or acatalytic domain of the polypeptide. In certain further embodiments ofthe method, the polypeptide of the method is derived from a toxin. Incertain further embodiments of the method, the polypeptide comprises atoxin effector polypeptide capable of intracellular delivery of a T-cellepitope from an early endosomal compartment to a proteasome of a cell inwhich the toxin effector polypeptide is present, and the methodcomprises embedding or inserting the heterologous T-cell epitope in thetoxin effector polypeptide. In certain further embodiments of themethod, the embedding or inserting step results in a toxin effectorpolypeptide capable of exhibiting one or more toxin effector functionsin addition to intracellular delivery of a T-cell epitope from an earlyendosomal compartment to a MHC class I molecule of a cell in which thetoxin effector polypeptide is present.

In certain embodiments of the methods of the present invention is amethod of increasing CD8+ T-cell immunogenicity of a polypeptide capableof intracellular delivery of a T-cell epitope from an early endosomalcompartment to a proteasome of a cell in which the polypeptide ispresent, the method comprising the step of: embedding or inserting aheterologous CD8+ T-cell epitope in the polypeptide. In certain furtherembodiments of the method, the polypeptide of the method is derived froma toxin. In certain further embodiments of the method, the polypeptidecomprises a toxin effector polypeptide capable of intracellular deliveryof a T-cell epitope from an early endosomal compartment to a proteasomeof a cell in which the toxin effector polypeptide is present, and themethod comprises embedding or inserting the heterologous T-cell epitopein the toxin effector polypeptide. In certain further embodiments of themethod, the embedding or inserting step results in a toxin effectorpolypeptide capable of exhibiting one or more toxin effector functionsin addition to intracellular delivery of a T-cell epitope from an earlyendosomal compartment to a MHC class I molecule of a cell in which thetoxin effector polypeptide is present.

In certain embodiments of the methods of the present invention is amethod of increasing CD8+ T-cell immunogenicity of a polypeptide capableof intracellular delivery of a T-cell epitope from an early endosomalcompartment to a MHC class I molecule of a cell in which the polypeptideis present, the method comprising the step of: embedding or inserting aheterologous CD8+ T-cell epitope in the polypeptide. In certain furtherembodiments of the method, the polypeptide of the method is derived froma toxin. In certain further embodiments of the method, the polypeptidecomprises a toxin effector polypeptide capable of intracellular deliveryof a T-cell epitope from an early endosomal compartment to a proteasomeof a cell in which the toxin effector polypeptide is present, and themethod comprises embedding or inserting the heterologous T-cell epitopein the toxin effector polypeptide. In certain further embodiments of themethod, the embedding or inserting step results in a toxin effectorpolypeptide capable of exhibiting one or more toxin effector functionsin addition to intracellular delivery of a T-cell epitope from an earlyendosomal compartment to a MHC class I molecule of a cell in which thetoxin effector polypeptide is present.

In certain embodiments of the methods of the present invention is amethod of creating a T-cell epitope delivery molecule capable ofintracellular delivery of a T-cell epitope from an early endosomalcompartment to the cytosol, endoplasmic reticulum, and/or lysosome of acell in which the molecule is present, the method comprising the stepof: associating a heterologous T-cell epitope with a polypeptide capableof routing to a subcellular compartment of a cell in which thepolypeptide is present selected from the group consisting of: cytosol,endoplasmic reticulum, and lysosome. In certain further embodiments ofthe method, the associating consists of embedding or inserting theheterologous T-cell epitope in an endogenous B-cell epitope, anendogenous CD4+ T-cell epitope, and/or a catalytic domain of themolecule. In certain further embodiments of the method, the polypeptideof the method is derived from a toxin. In certain further embodiments ofthe method, the polypeptide comprises a toxin effector polypeptidecapable of intracellular delivery of a T-cell epitope from an earlyendosomal compartment to the cytosol, endoplasmic reticulum, and/orlysosome of a cell in which the toxin effector polypeptide is present,and the method comprises embedding or inserting the heterologous T-cellepitope in the toxin effector polypeptide. In certain furtherembodiments of the method, the embedding or inserting step results in atoxin effector polypeptide capable of exhibiting one or more toxineffector functions in addition to intracellular delivery of a T-cellepitope from an early endosomal compartment to the cytosol, endoplasmicreticulum, and/or lysosome of a cell in which the toxin effectorpolypeptide is present.

In certain embodiments of the methods of the present invention is amethod of creating a CD8+ T-cell epitope delivery molecule capable ofintracellular delivery of a T-cell epitope from an early endosomalcompartment to a proteasome of a cell in which the delivery molecule ispresent, the method comprising the step of: embedding or inserting aheterologous CD8+ T-cell epitope in a proteasome delivering effectorpolypeptide capable of intracellular delivery of a T-cell epitope froman early endosomal compartment to a proteasome of a cell in which theproteasome delivering effector polypeptide is present. In certainfurther embodiments of the method, the associating consists of embeddingor inserting the heterologous T-cell epitope in an endogenous B-cellepitope, an endogenous CD4+ T-cell epitope, and/or a catalytic domain ofthe molecule. In certain further embodiments of the method, thepolypeptide of the method is derived from a toxin. In certain furtherembodiments of the method, the polypeptide comprises a toxin effectorpolypeptide capable of exhibiting one or more toxin effector functionsin addition to intracellular delivery of a T-cell epitope from an earlyendosomal compartment to a proteasome of a cell in which the toxineffector polypeptide is present.

In certain embodiments of the methods of the present invention is amethod of creating a CD8+ T-cell epitope delivery molecule capable ofintracellular delivery of a T-cell epitope from an early endosomalcompartment to a MHC class I molecule of a cell in which the deliverymolecule is present, the method comprising the step of: embedding orinserting a heterologous CD8+ T-cell epitope in a proteasome deliveringeffector polypeptide capable of intracellular delivery of a T-cellepitope from an early endosomal compartment to a MHC class I molecule ofa cell in which the proteasome delivering effector polypeptide ispresent. In certain further embodiments of the method, the associatingconsists of embedding or inserting the heterologous T-cell epitope in anendogenous B-cell epitope, an endogenous CD4+ T-cell epitope, and/or acatalytic domain of the molecule. In certain further embodiments of themethod, the polypeptide of the method is derived from a toxin. Incertain further embodiments of the method, the polypeptide comprises atoxin effector polypeptide comprising the proteasome delivering effectorpolypeptide, and the method comprises embedding or inserting theheterologous T-cell epitope in the toxin effector polypeptide. Incertain further embodiments of the method, the toxin effectorpolypeptide resulting from the is capable of exhibiting one or moretoxin effector functions in addition to intracellular delivery of aT-cell epitope from an early endosomal compartment to a MHC class Imolecule of a cell in which the toxin effector polypeptide is present.

In certain embodiments of the methods of the present invention is amethod of creating a CD8+ T-cell epitope delivery molecule capable whenpresent in a cell of delivering a T-cell epitope for presentation by aMHC class I molecule, the method comprising the step of: embedding orinserting a heterologous CD8+ T-cell epitope in a proteasome deliveringeffector polypeptide capable of intracellular delivery of a T-cellepitope from an early endosomal compartment to a proteasome of a cell inwhich the proteasome delivering effector polypeptide is present. Incertain further embodiments of the method, the associating consists ofembedding or inserting the heterologous T-cell epitope in an endogenousB-cell epitope, an endogenous CD4+ T-cell epitope, and/or a catalyticdomain of the molecule. In certain further embodiments of the method,the polypeptide of the method is derived from a toxin. In certainfurther embodiments of the method, the polypeptide comprises a toxineffector polypeptide comprising the proteasome delivering effectorpolypeptide, and the method comprises embedding or inserting theheterologous T-cell epitope in the toxin effector polypeptide. Incertain further embodiments of the method, the toxin effectorpolypeptide resulting from the is capable of exhibiting one or moretoxin effector functions in addition to intracellular delivery of aT-cell epitope from an early endosomal compartment to a MHC class Imolecule of a cell in which the toxin effector polypeptide is present.

In certain embodiments of the methods of the present invention is amethod of creating a CD8+ T-cell epitope delivery molecule capable whenpresent in a cell of delivering a T-cell epitope for presentation by aMHC class I molecule, the method comprising the step of: embedding orinserting a heterologous CD8+ T-cell epitope in a proteasome deliveringeffector polypeptide capable of intracellular delivery of a T-cellepitope from an early endosomal compartment to a MHC class I molecule ofa cell in which the proteasome delivering effector polypeptide ispresent. In certain further embodiments of the method, the associatingconsists of embedding or inserting the heterologous T-cell epitope in anendogenous B-cell epitope, an endogenous CD4+ T-cell epitope, and/or acatalytic domain of the molecule. In certain further embodiments of themethod, the polypeptide of the method is derived from a toxin. Incertain further embodiments of the method, the polypeptide comprises atoxin effector polypeptide comprising the proteasome delivering effectorpolypeptide, and the method comprises embedding or inserting theheterologous T-cell epitope in the toxin effector polypeptide. Incertain further embodiments of the method, the toxin effectorpolypeptide resulting from the is capable of exhibiting one or moretoxin effector functions in addition to intracellular delivery of aT-cell epitope from an early endosomal compartment to a MHC class Imolecule of a cell in which the toxin effector polypeptide is present.

In certain embodiments, a de-immunized polypeptide of the presentinvention comprises a heterologous T-cell epitope disrupting anendogenous B-cell epitope and/or CD4+ T-cell epitope. In certain furtherembodiments, the polypeptide of the present invention comprises atoxin-derived polypeptide. In certain further embodiments, theheterologous CD8+ T-cell epitope is in the toxin-derived polypeptide. Incertain further embodiments, the toxin-derived polypeptide of thepresent invention comprises a toxin effector polypeptide. In certainfurther embodiments, the heterologous CD8+ T-cell epitope in the toxineffector polypeptide. In certain further embodiments, the toxin effectorpolypeptide is capable of exhibiting one or more toxin effectorfunctions. In certain further embodiments, the polypeptide of thepresent invention comprises the toxin effector polypeptide derived froma toxin selected from the group consisting of: ABx toxin, ribosomeinactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labileenterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein,pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin,sarcin, Shiga toxin, and subtilase cytotoxin. In certain furtherembodiments, the toxin effector polypeptide is a diphtheria toxineffector polypeptide comprising an amino acid sequence derived from theA and B Subunits of at least one member of the diphtheria toxin family,wherein the diphtheria toxin effector polypeptide comprises a disruptionof at least one B-cell epitope and/or CD4+ T-cell epitope region of theamino acid sequence selected from the group of natively positioned aminoacids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID NO:39, 71-77of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of SEQ ID NO:39,165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39; and wherein thediphtheria toxin effector polypeptide is capable of routing to a cytosolcompartment of a cell in which the diphtheria toxin effector polypeptideis present. In certain further embodiments, the polypeptide of thepresent invention comprises the diphtheria toxin effector polypeptidederived from amino acids 2 to 389 of SEQ ID NO:45. In certain furtherembodiment, the toxin effector polypeptide is a Shiga toxin effectorpolypeptide comprising an amino acid sequence derived from an A Subunitof at least one member of the Shiga toxin family, wherein the Shigatoxin effector polypeptide comprises a disruption of at least one B-cellepitope and/or CD4+ T-cell epitope region of the Shiga toxin A Subunitamino acid sequence selected from the group of natively positioned aminoacids consisting of: the B-cell epitope regions 1-15 of SEQ ID NO:1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ IDNO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQID NO:2; and 210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 ofSEQ ID NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2;262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ IDNO:1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of SEQ IDNO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO: 1 or SEQ ID NO:2, 77-103 ofSEQ ID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ ID NO:2,160-183 of SEQ ID NO: 1 or SEQ ID NO:2, 236-258 of SEQ ID NO: 1 or SEQID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; and wherein theShiga toxin effector polypeptide is capable of routing to a cytosolcompartment of a cell in which the Shiga toxin effector polypeptide ispresent. In certain embodiments, the polypeptide of the presentinvention comprises the Shiga toxin effector polypeptide derived fromamino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. Incertain further embodiments, the polypeptide of the present inventioncomprises the Shiga toxin effector polypeptide derived from amino acids1 to 241 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certainfurther embodiments, the Shiga toxin effector polypeptide is derivedfrom amino acids 1 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.In certain further embodiments, the Shiga toxin effector polypeptide isderived from amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3.

In certain embodiments, a polypeptide of the present invention comprisesa heterologous CD8+ T-cell epitope disrupting an endogenous B-cellepitope and/or an endogenous CD4+ T-cell epitope, wherein thepolypeptide is capable of intracellular delivery of the CD8+ T-cellepitope from an early endosomal compartment to a proteasome of a cell inwhich the polypeptide is present. In certain further embodiments, thepolypeptide of the present invention comprises a toxin-derivedpolypeptide. In certain further embodiments, the heterologous CD8+T-cell epitope is in the toxin-derived polypeptide. In certain furtherembodiments, the toxin-derived polypeptide of the present inventioncomprises a toxin effector polypeptide. In certain further embodiments,the heterologous CD8+ T-cell epitope in the toxin effector polypeptide.In certain further embodiments, the toxin effector polypeptide iscapable of exhibiting one or more toxin effector functions. In certainfurther embodiments, the polypeptide of the present invention comprisesthe toxin effector polypeptide derived from a toxin selected from thegroup consisting of: ABx toxin, ribosome inactivating protein toxin,abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin,diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin,pertussis toxin, pokeweed antiviral protein, pulchellin, Pseudomonasexotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, andsubtilase cytotoxin. In certain further embodiments, the toxin effectorpolypeptide is a diphtheria toxin effector polypeptide comprising anamino acid sequence derived from the A and B Subunits of at least onemember of the diphtheria toxin family, wherein the diphtheria toxineffector polypeptide comprises a disruption of at least one B-cellepitope and/or CD4+ T-cell epitope region of the amino acid sequenceselected from the group of natively positioned amino acids consistingof: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID NO:39, 71-77 of SEQ ID NO:39,125-131 of SEQ ID NO:39, 138-146 of SEQ ID NO:39, 165-175 of SEQ IDNO:39, and 185-191 of SEQ ID NO:39; and wherein the diphtheria toxineffector polypeptide is capable of routing to a cytosol compartment of acell in which the diphtheria toxin effector polypeptide is present. Incertain further embodiments, the polypeptide of the present inventioncomprises the diphtheria toxin effector polypeptide derived from aminoacids 2 to 389 of SEQ ID NO:45. In certain further embodiment, the toxineffector polypeptide is a Shiga toxin effector polypeptide comprising anamino acid sequence derived from an A Subunit of at least one member ofthe Shiga toxin family, wherein the Shiga toxin effector polypeptidecomprises a disruption of at least one B-cell epitope and/or CD4+ T-cellepitope region of the Shiga toxin A Subunit amino acid sequence selectedfrom the group of natively positioned amino acids consisting of: theB-cell epitope regions 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2;39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 ofSEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ IDNO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ IDNO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3;281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2, andthe CD4+ T-cell epitope regions 4-33 of SEQ ID NO:1 or SEQ ID NO:2,34-78 of SEQ ID NO:1 or SEQ ID NO:2, 77-103 of SEQ ID NO:1 or SEQ IDNO:2, 128-168 of SEQ ID NO:1 or SEQ ID NO:2, 160-183 of SEQ ID NO:1 orSEQ ID NO:2, 236-258 of SEQ ID NO:1 or SEQ ID NO:2, and 274-293 of SEQID NO: 1 or SEQ ID NO:2; and wherein the Shiga toxin effectorpolypeptide is capable of routing to a cytosol compartment of a cell inwhich the Shiga toxin effector polypeptide is present. In certainembodiments, the polypeptide of the present invention comprises theShiga toxin effector polypeptide derived from amino acids 75 to 251 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain furtherembodiments, the polypeptide of the present invention comprises theShiga toxin effector polypeptide derived from amino acids 1 to 241 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain furtherembodiments, the Shiga toxin effector polypeptide is derived from aminoacids 1 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certainfurther embodiments, the Shiga toxin effector polypeptide is derivedfrom amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

In certain embodiments, a de-immunized polypeptide of the presentinvention comprises a heterologous CD8+ T-cell epitope disrupting anendogenous B-cell epitope and/or CD4+ T-cell epitope, wherein thepolypeptide is capable of intracellular delivery of the CD8+ T-cellepitope to a MHC class I molecule from an early endosomal compartment ofa cell in which the polypeptide is present. In certain furtherembodiments, the polypeptide of the present invention comprises atoxin-derived polypeptide. In certain further embodiments, theheterologous CD8+ T-cell epitope is in the toxin-derived polypeptide. Incertain further embodiments, the toxin-derived polypeptide of thepresent invention comprises a toxin effector polypeptide. In certainfurther embodiments, the heterologous CD8+ T-cell epitope in the toxineffector polypeptide. In certain further embodiments, the toxin effectorpolypeptide is capable of exhibiting one or more toxin effectorfunctions. In certain further embodiments, the polypeptide of thepresent invention comprises the toxin effector polypeptide derived froma toxin selected from the group consisting of: ABx toxin, ribosomeinactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labileenterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein,pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin,sarcin, Shiga toxin, and subtilase cytotoxin. In certain furtherembodiments, the toxin effector polypeptide is a diphtheria toxineffector polypeptide comprising an amino acid sequence derived from theA and B Subunits of at least one member of the diphtheria toxin family,wherein the diphtheria toxin effector polypeptide comprises a disruptionof at least one B-cell epitope and/or CD4+ T-cell epitope region of theamino acid sequence selected from the group of natively positioned aminoacids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID NO:39, 71-77of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of SEQ ID NO:39,165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39; and wherein thediphtheria toxin effector polypeptide is capable of routing to a cytosolcompartment of a cell in which the diphtheria toxin effector polypeptideis present. In certain further embodiments, the polypeptide of thepresent invention comprises the diphtheria toxin effector polypeptidederived from amino acids 2 to 389 of SEQ ID NO:45. In certain furtherembodiment, the toxin effector polypeptide is a Shiga toxin effectorpolypeptide comprising an amino acid sequence derived from an A Subunitof at least one member of the Shiga toxin family, wherein the Shigatoxin effector polypeptide comprises a disruption of at least one B-cellepitope and/or CD4+ T-cell epitope region of the Shiga toxin A Subunitamino acid sequence selected from the group of natively positioned aminoacids consisting of: the B-cell epitope regions 1-15 of SEQ ID NO:1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ IDNO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQID NO:2; and 210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 ofSEQ ID NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2;262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ IDNO: 1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of SEQ IDNO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO: 1 or SEQ ID NO:2, 77-103 ofSEQ ID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ ID NO:2,160-183 of SEQ ID NO: 1 or SEQ ID NO:2, 236-258 of SEQ ID NO: 1 or SEQID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; and wherein theShiga toxin effector polypeptide is capable of routing to a cytosolcompartment of a cell in which the Shiga toxin effector polypeptide ispresent. In certain embodiments, the polypeptide of the presentinvention comprises the Shiga toxin effector polypeptide derived fromamino acids 75 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. Incertain further embodiments, the polypeptide of the present inventioncomprises the Shiga toxin effector polypeptide derived from amino acids1 to 241 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certainfurther embodiments, the Shiga toxin effector polypeptide is derivedfrom amino acids 1 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.In certain further embodiments, the Shiga toxin effector polypeptide isderived from amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO:2, or SEQID NO:3.

In certain embodiments, a de-immunized polypeptide of the presentinvention comprises a heterologous CD8+ T-cell epitope disrupting anendogenous B-cell epitope and/or CD4+ T-cell epitope, wherein thepolypeptide is capable of intracellular delivery of the CD 8+ T-cellepitope for presentation by a MHC class I molecule on the surface of acell in which the polypeptide is present. In certain furtherembodiments, the polypeptide of the present invention comprises atoxin-derived polypeptide. In certain further embodiments, theheterologous CD8+ T-cell epitope is in the toxin-derived polypeptide. Incertain further embodiments, the toxin-derived polypeptide of thepresent invention comprises a toxin effector polypeptide. In certainfurther embodiments, the heterologous CD8+ T-cell epitope in the toxineffector polypeptide. In certain further embodiments, the toxin effectorpolypeptide is capable of exhibiting one or more toxin effectorfunctions. In certain further embodiments, the polypeptide of thepresent invention comprises the toxin effector polypeptide derived froma toxin selected from the group consisting of: ABx toxin, ribosomeinactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labileenterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein,pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin,sarcin, Shiga toxin, and subtilase cytotoxin. In certain furtherembodiments, the toxin effector polypeptide is a diphtheria toxineffector polypeptide comprising an amino acid sequence derived from theA and B Subunits of at least one member of the diphtheria toxin family,wherein the diphtheria toxin effector polypeptide comprises a disruptionof at least one B-cell epitope and/or CD4+ T-cell epitope region of theamino acid sequence selected from the group of natively positioned aminoacids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID NO:39, 71-77of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of SEQ ID NO:39,165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39; and wherein thediphtheria toxin effector polypeptide is capable of routing to a cytosolcompartment of a cell in which the diphtheria toxin effector polypeptideis present. In certain further embodiments, the polypeptide of thepresent invention comprises the diphtheria toxin effector polypeptidederived from amino acids 2 to 389 of SEQ ID NO:45. In certain furtherembodiment, the toxin effector polypeptide is a Shiga toxin effectorpolypeptide comprising an amino acid sequence derived from an A Subunitof at least one member of the Shiga toxin family, wherein the Shigatoxin effector polypeptide comprises a disruption of at least one B-cellepitope and/or CD4+ T-cell epitope region of the Shiga toxin A Subunitamino acid sequence selected from the group of natively positioned aminoacids consisting of: the B-cell epitope regions 1-15 of SEQ ID NO:1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ IDNO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQID NO:2; and 210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 ofSEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2;262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ IDNO:1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of SEQ IDNO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO:1 or SEQ ID NO:2, 77-103 of SEQID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ ID NO:2, 160-183of SEQ ID NO:1 or SEQ ID NO:2, 236-258 of SEQ ID NO:1 or SEQ ID NO:2,and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; and wherein the Shiga toxineffector polypeptide is capable of routing to a cytosol compartment of acell in which the Shiga toxin effector polypeptide is present. Incertain embodiments, the polypeptide of the present invention comprisesthe Shiga toxin effector polypeptide derived from amino acids 75 to 251of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain furtherembodiments, the polypeptide of the present invention comprises theShiga toxin effector polypeptide derived from amino acids 1 to 241 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain furtherembodiments, the Shiga toxin effector polypeptide is derived from aminoacids 1 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certainfurther embodiments, the Shiga toxin effector polypeptide is derivedfrom amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

In certain embodiments, a de-immunized polypeptide of the presentinvention comprises a proteasome delivering effector polypeptidecomprising a first heterologous T-cell epitope disrupting an endogenousB-cell epitope and/or CD4+ T-cell epitope, wherein the proteasomedelivering effector polypeptide is linked to a second CD8+ T-cellepitope; and the polypeptide is capable of intracellular delivery of thesecond CD8+ T-cell epitope for presentation by a MHC class I molecule onthe surface of a cell in which the polypeptide is present. In certainfurther embodiments, the polypeptide of the present invention comprisesa toxin-derived polypeptide. In certain further embodiments, theheterologous CD8+ T-cell epitope is in the toxin-derived polypeptide. Incertain further embodiments, the toxin-derived polypeptide of thepresent invention comprises a toxin effector polypeptide. In certainfurther embodiments, the heterologous CD8+ T-cell epitope in the toxineffector polypeptide. In certain further embodiments, the toxin effectorpolypeptide is capable of exhibiting one or more toxin effectorfunctions. In certain further embodiments, the polypeptide of thepresent invention comprises the toxin effector polypeptide derived froma toxin selected from the group consisting of: ABx toxin, ribosomeinactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin,bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labileenterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein,pulchellin, Pseudomonas exotoxin A, restrictocin, ricin, saporin,sarcin, Shiga toxin, and subtilase cytotoxin. In certain furtherembodiments, the toxin effector polypeptide is a diphtheria toxineffector polypeptide comprising an amino acid sequence derived from theA and B Subunits of at least one member of the diphtheria toxin family,wherein the diphtheria toxin effector polypeptide comprises a disruptionof at least one B-cell epitope and/or CD4+ T-cell epitope region of theamino acid sequence selected from the group of natively positioned aminoacids consisting of: 3-10 of SEQ ID NO:39, 33-43 of SEQ ID NO:39, 71-77of SEQ ID NO:39, 125-131 of SEQ ID NO:39, 138-146 of SEQ ID NO:39,165-175 of SEQ ID NO:39, and 185-191 of SEQ ID NO:39; and wherein thediphtheria toxin effector polypeptide is capable of routing to a cytosolcompartment of a cell in which the diphtheria toxin effector polypeptideis present. In certain further embodiments, the polypeptide of thepresent invention comprises the diphtheria toxin effector polypeptidederived from amino acids 2 to 389 of SEQ ID NO:45. In certain furtherembodiment, the toxin effector polypeptide is a Shiga toxin effectorpolypeptide comprising an amino acid sequence derived from an A Subunitof at least one member of the Shiga toxin family, wherein the Shigatoxin effector polypeptide comprises a disruption of at least one B-cellepitope and/or CD4+ T-cell epitope region of the Shiga toxin A Subunitamino acid sequence selected from the group of natively positioned aminoacids consisting of: the B-cell epitope regions 1-15 of SEQ ID NO:1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ IDNO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO: 1 or SEQID NO:2; and 210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 ofSEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2;262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ IDNO:1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of SEQ IDNO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO:1 or SEQ ID NO:2, 77-103 of SEQID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ ID NO:2, 160-183of SEQ ID NO:1 or SEQ ID NO:2, 236-258 of SEQ ID NO:1 or SEQ ID NO:2,and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; and wherein the Shiga toxineffector polypeptide is capable of routing to a cytosol compartment of acell in which the Shiga toxin effector polypeptide is present. Incertain embodiments, the polypeptide of the present invention comprisesthe Shiga toxin effector polypeptide derived from amino acids 75 to 251of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain furtherembodiments, the polypeptide of the present invention comprises theShiga toxin effector polypeptide derived from amino acids 1 to 241 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certain furtherembodiments, the Shiga toxin effector polypeptide is derived from aminoacids 1 to 251 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In certainfurther embodiments, the Shiga toxin effector polypeptide is derivedfrom amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

In certain embodiments of the methods of the present invention is amethod for reducing B-cell immunogenicity in a polypeptide, the methodcomprising the step of: disrupting a B-cell epitope with one or moreamino acid residue(s) of a T-cell epitope added to the polypeptide. Incertain further embodiments, the disrupting step further comprises thestep or steps of making one or more amino acid substitutions in theB-cell epitope. In certain further embodiments, the disrupting stepfurther comprises the step or steps of making one or more amino acidinsertions in the B-cell epitope.

In certain embodiments of the methods of the present invention is amethod for reducing B-cell immunogenicity in a polypeptide, the methodcomprising the steps of: identifying a B-cell epitope in a polypeptide;and disrupting the identified B-cell epitope with one or more amino acidresidue(s) in a T-cell epitope added to polypeptide. In certain furtherembodiments, the disrupting step further comprises the step or steps ofmaking one or more amino acid substitutions in the B-cell epitope. Incertain further embodiments, the disrupting step further comprises thestep or steps of making one or more amino acid insertions in the B-cellepitope.

In certain embodiments of the methods of the present invention is amethod for reducing B-cell immunogenicity in a polypeptide whilesimultaneously increasing CD8+ T-cell immunogenicity of the polypeptide,the method comprising the step of: disrupting a B-cell epitope with oneor more amino acid residue(s) in a heterologous CD8+ T-cell epitopeadded to the polypeptide. In certain further embodiments, the disruptingstep further comprises the step or steps of making one or more aminoacid substitutions in the B-cell epitope. In certain furtherembodiments, the disrupting step further comprises the step or steps ofmaking one or more amino acid insertions in the B-cell epitope.

In certain embodiments of the methods of the present invention is amethod for reducing B-cell immunogenicity in a polypeptide whilesimultaneously increasing CD8+ T-cell immunogenicity of the polypeptide,the method comprising the steps of: identifying a CD4+ T-cell epitope ina polypeptide; and disrupting the identified CD4+ T-cell epitope withone or more amino acid residue(s) in a CD8+ T-cell epitope added to thepolypeptide. In certain further embodiments, the disrupting step furthercomprises the step or steps of making one or more amino acidsubstitutions in the B-cell epitope. In certain further embodiments, thedisrupting step further comprises the step or steps of making one ormore amino acid insertions in the B-cell epitope.

In certain embodiments of the methods of the present invention is methodfor reducing CD4+ T-cell immunogenicity in a polypeptide, the methodcomprising the step of: disrupting a CD4+ T-cell epitope with one ormore amino acid residue(s) in a CD8+ T-cell epitope added to thepolypeptide. In certain further embodiments, the disrupting step furthercomprises the step or steps of making one or more amino acidsubstitutions in the CD4+ T-cell epitope. In certain furtherembodiments, the disrupting step further comprises the step or steps ofmaking one or more amino acid insertions in the CD4+ T-cell epitope.

In certain embodiments of the methods of the present invention is amethod for reducing CD4+ T-cell immunogenicity in a polypeptide, themethod comprising the steps of: identifying a CD4+ T-cell epitope in apolypeptide; and disrupting the identified CD4+ T-cell epitope with oneor more amino acid residue(s) in a CD8+ T-cell epitope added to thepolypeptide. In certain further embodiments, the disrupting step furthercomprises the step or steps of making one or more amino acidsubstitutions in the CD4+ T-cell epitope. In certain furtherembodiments, the disrupting step further comprises the step or steps ofmaking one or more amino acid insertions in the CD4+ T-cell epitope.

In certain embodiments of the methods of the present invention is amethod for reducing CD4+ T-cell immunogenicity in a polypeptide whilesimultaneously increasing CD8+ T-cell immunogenicity of the polypeptide,the method comprising the step of: disrupting a CD4+ T-cell epitope withone or more amino acid residue(s) in a heterologous CD8+ T-cell epitopeadded to the polypeptide. In certain further embodiments, the disruptingstep further comprises the step or steps of making one or more aminoacid substitutions in the CD4+ T-cell epitope. In certain furtherembodiments, the disrupting step further comprises the step or steps ofmaking one or more amino acid insertions in the CD4+ T-cell epitope.

In certain embodiments of the methods of the present invention is amethod for reducing CD4+ T-cell immunogenicity in a polypeptide whilesimultaneously increasing CD8+ T-cell immunogenicity of the polypeptide,the method comprising the steps of: identifying a CD4+ T-cell epitope ina polypeptide; and disrupting the identified CD4+ T-cell epitope withone or more amino acid residue(s) in a CD8+ T-cell epitope added to thepolypeptide. In certain further embodiments, the disrupting step furthercomprises the step or steps of making one or more amino acidsubstitutions in the CD4+ T-cell epitope. In certain furtherembodiments, the disrupting step further comprises the step or steps ofmaking one or more amino acid insertions in the CD4+ T-cell epitope.

Certain embodiments of the polypeptides of the present invention providea polypeptide produced by any of the methods of the present invention.

In certain embodiments, the polypeptide of the present inventioncomprises or consists essentially of any one of SEQ ID NOs: 11-43 or46-48.

In certain embodiments, a cell-targeted molecule of the presentinvention comprises a cell-targeting moiety or agent, and anypolypeptide of the present invention. In certain further embodiments,the cell-targeted molecule further comprises a binding region comprisingone or more polypeptides and capable of specifically binding at leastone extracellular target biomolecule. In certain further embodiments,the binding region comprises a polypeptide selected from the groupconsisting of: a complementary determining region 3 (CDR3) fragmentconstrained FR3-CDR3-FR4 (FR3-CDR3-FR4) polypeptide, single-domainantibody fragment (sdAb), nanobody, heavy-chain antibody domain derivedfrom a camelid (V_(H)H fragment), heavy-chain antibody domain derivedfrom a cartilaginous fish, immunoglobulin new antigen receptors(IgNARs), V_(NAR) fragment, single-chain variable fragment (scFv),antibody variable fragment (Fv), antigen-binding fragment (Fab), Fdfragment, small modular immunopharmaceutical (SMIP) domain,fibronectin-derived 10^(th) fibronectin type III domain (10Fn3) (e.g.monobody), tenascin type III domain (e.g. TNfn3), ankyrin repeat motifdomain (ARD), low-density-lipoprotein-receptor-derived A-domain (Adomain of LDLR or LDLR-A), lipocalin (anticalin), Kunitz domain,Protein-A-derived Z domain, gamma-B crystalline-derived domain(Affilin), ubiquitin-derived domain, Sac7d-derived polypeptide,Fyn-derived SH2 domain (affitin), miniprotein, C-type lectin-like domainscaffold, engineered antibody mimic, and any genetically manipulatedcounterparts of any of the foregoing that retain binding functionality.In certain further embodiments of the cell-targeted molecule of thepresent invention, whereby upon administration of the cell-targetedmolecule to a cell physically coupled with an extracellular targetbiomolecule of the binding region, the cell-targeted molecule is capableof causing death of the cell. In certain further embodiments of thecell-targeted molecule of the present invention, whereby uponadministration of the cell-targeted molecule to a first population ofcells whose members are physically coupled to extracellular targetbiomolecules of the binding region, and a second population of cellswhose members are not physically coupled to any extracellular targetbiomolecule of said binding region, the cytotoxic effect of thecell-targeted molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. In certain further embodiments of the cell-targetedmolecules of the present invention, the binding region is capable ofbinding to an extracellular target biomolecule selected from the groupconsisting of: CD20, CD22, CD40, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR,EpCAM, EphB2, prostate-specific membrane antigen, Cripto, endoglin,fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33,CD52, EpCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase IX, folatebinding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2,ganglioside Lewis-Y2, VEGFR, Alpha Vbeta3, Alpha5beta1, ErbB1/EGFR,Erb3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, tenascin,CD64, mesothelin, BRCA1, MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2,MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-catenin,MUM-1, caspase-8, KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonicantigen, prostate specific antigen, prostate stem cell antigen, humanaspartyl (asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1,MART-1/MelanA, gp100, tyrosinase associated antigen, HPV-E7,Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen, 17-Al,bladder tumor antigen, CD38, CD15, CD23, CD53, CD88, CD129, CD183,CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa, galectin-9, mrp-14,Siglec-8, Siglec-10, CD49d, CD13, CD44, CD54, CD63, CD69, CD123, TLR4,FceRIa, IgE, CD107a, CD203c, CD14, CD68, CD80, CD86, CD105, CD115,F4/80, ILT-3, galectin-3, CD11a-c, GITRL, MHC Class II, CD284-TLR4,CD107-Mac3, CD195-CCR5, HLA-DR, CD16/32, CD282-TLR2, CD11c, and anyimmunogenic fragment of any of the foregoing. In certain furtherembodiments of the cell-targeted molecules of the present invention, thecell-targeted molecule further comprises a carboxy-terminal endoplasmicreticulum retention/retrieval signal motif of a member of the KDELfamily. In certain further embodiments, the carboxy-terminal endoplasmicreticulum retention/retrieval signal motif selected from the groupconsisting of: KDEL, HDEF, HDEL, RDEF, RDEL, WDEL, YDEL, HEEF, HEEL,KEEL, REEL, KAEL, KCEL, KFEL, KGEL, KHEL, KLEL, KNEL, KQEL, KREL, KSEL,KVEL, KWEL, KYEL, KEDL, KIEL, DKEL, FDEL, KDEF, KKEL, HADL, HAEL, HIEL,HNEL, HTEL, KTEL, HVEL, NDEL, QDEL, REDL, RNEL, RTDL, RTEL, SDEL, TDEL,and SKEL.

In certain embodiments of the present invention, upon administration ofthe cell-targeted molecule of the present invention to a cell physicallycoupled with an extracellular target biomolecule of the cell-targetingmoiety of the cytotoxic protein, the cytotoxic protein is capable ofcausing death of the cell.

In certain embodiments of the present invention, upon administration ofthe cell-targeted molecule of the present invention to two differentpopulations of cell types with respect to the presence of anextracellular target biomolecule, the cell-targeted molecule is capableof causing cell death to the cell-types physically coupled with anextracellular target biomolecule of the cell-targeting moiety or agent'sbinding region at a CD₅₀ at least three times or less than the CD₅₀ tocell types which are not physically coupled with an extracellular targetbiomolecule of the cell-targeted molecule's cell-targeting moiety.

In certain embodiments, the cell-targeted molecule of the presentinvention comprises or consists essentially of a polypeptide of any oneof the amino acid sequences of SEQ ID NOs: 49-60.

In certain further embodiments, the polypeptides of the presentinvention comprise a mutation which reduces or eliminates catalyticactivity of a toxin-derived polypeptide but retains at least one othertoxin effector function. In certain embodiments, the cell-targetedmolecule of the present invention further comprises a toxin effectorpolypeptide, derived from a toxin effector polypeptide with enzymaticactivity, which comprises a mutation relative to a naturally occurringtoxin which changes the enzymatic activity of the toxin effectorpolypeptide. In certain further embodiments, the mutation is selectedfrom at least one amino acid residue deletion, insertion, orsubstitution that reduces or eliminates cytotoxicity of the toxineffector polypeptide. In certain embodiments, the cell-targetedmolecules of the invention comprise a Shiga toxin effector region whichfurther comprises a mutation relative to a naturally occurring A Subunitof a member of the Diphtheria toxin family that changes the enzymaticactivity of the diphtheria toxin effector region, the mutation selectedfrom at least one amino acid residue deletion or substitution, such as,e.g. H21A, Y27A, W50A, Y54A, Y65A, E148A, and W153A. In certainembodiments, the cell-targeted molecules of the invention comprise aShiga toxin effector region which further comprises a mutation relativeto a naturally occurring A Subunit of a member of the Shiga toxin familythat changes the enzymatic activity of the Shiga toxin effector region,the mutation selected from at least one amino acid residue deletion orsubstitution, such as, e.g., A231E, R75A, Y77S, Y114S, E167D, R170A,R176K and/or W203A in SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.

The present invention also provides pharmaceutical compositionscomprising a polypeptide and/or cell-targeted molecule of the inventionand at least one pharmaceutically acceptable excipient or carrier; andthe use of such a polypeptide, cell-targeted molecule, or a compositioncomprising it in methods of the invention as further described herein.Certain embodiments of the present invention are pharmaceuticalcompositions comprising any polypeptide of the present invention and/orany cell-targeted molecule of the present invention; and at least onepharmaceutically acceptable excipient or carrier.

Beyond the polypeptides, cell-targeted molecules, proteins, andcompositions of the present invention, polynucleotides capable ofencoding a polypeptide comprising a polypeptide or cell-targetedmolecule or protein of the present invention comprising a polypeptide ofthe invention are within the scope of the present invention, as well asexpression vectors which comprise a polynucleotide of the invention andhost cells comprising an expression vector of the invention. Host cellscomprising an expression vector may be used, e.g., in methods forproducing a polypeptide and/or protein of the invention comprising it,or a polypeptide component or fragment thereof, by recombinantexpression.

Additionally, the present invention provides methods of selectivelykilling cell(s) comprising the step of contacting a cell(s) with acell-targeted molecule of the invention or a pharmaceutical compositioncomprising such a protein of the invention. In certain embodiments, thestep of contacting the cell(s) occurs in vitro. In certain otherembodiments, the step of contacting the cell(s) occurs in vivo.

The present invention further provides methods of treating diseases,disorders, and/or conditions in patients in need thereof comprising thestep of administering to a patient in need thereof a therapeuticallyeffective amount of a composition comprising a polypeptide of theinvention, a polypeptide and/or protein comprising it, or a compositioncomprising any of the foregoing (e.g., a pharmaceutical composition). Incertain embodiments, the disease, disorder, or condition to be treatedusing this method of the invention is selected from: a cancer, tumor,immune disorder, or microbial infection. In certain embodiments of thismethod, the cancer to be treated is selected from the group consistingof: bone cancer, breast cancer, central/peripheral nervous systemcancer, gastrointestinal cancer, germ cell cancer, glandular cancer,head-neck cancer, hematological cancer, kidney-urinary tract cancer,liver cancer, lung/pleura cancer, prostate cancer, sarcoma, skin cancer,and uterine cancer. In certain embodiments of this method, the immunedisorder to be treated is an immune disorder associated with a diseaseselected from the group consisting of: amyloidosis, ankylosingspondylitis, asthma, Crohn's disease, diabetes, graft rejection,graft-versus-host disease, Hashimoto's thyroiditis, hemolytic uremicsyndrome, HIV-related diseases, lupus erythematosus, multiple sclerosis,polyarteritis, psoriasis, psoriatic arthritis, rheumatoid arthritis,scleroderma, septic shock, Sjorgren's syndrome, ulcerative colitis, andvasculitis.

Among certain embodiments of the present invention is a compositioncomprising a polypeptide of the invention, a polypeptide and/orcell-targeted molecule comprising it, or a composition comprising any ofthe foregoing, for the treatment or prevention of a cancer, tumor,immune disorder, or microbial infection. Among certain embodiments ofthe present invention is the use of a composition of matter of theinvention in the manufacture of a medicament for the treatment orprevention of a cancer, tumor, immune disorder, or microbial infection.

Certain embodiments of the cell-targeted molecules of the presentinvention may be used to deliver one or more additional exogenousmaterials into a cell physically coupled with an extracellular targetbiomolecule of the protein of the present invention. Additionally, thepresent invention provides a method for delivering exogenous material tothe inside of a cell(s) comprising contacting the cell(s), either invitro or in vivo, with a cell-targeted molecule, pharmaceuticalcomposition, and/or diagnostic composition of the present invention. Thepresent invention further provides a method for delivering exogenousmaterial to the inside of a cell(s) in a patient in need thereof, themethod comprising the step of administering to the patient acell-targeted molecule of the present invention, wherein the targetcell(s) is physically coupled with an extracellular target biomoleculeof the protein of the present invention.

Among certain embodiments of the present invention is the use of acompound (e.g. a polypeptide or a cell-targeted molecule) of theinvention and/or composition (e.g. a pharmaceutical composition) of theinvention in the diagnosis, prognosis, or characterization of a disease,disorder, or condition.

Among certain embodiments of the present invention is a diagnosticcomposition comprising a polypeptide of the invention and/orcell-targeted molecule comprising it, or a composition comprising any ofthe foregoing, and a detection promoting agent for the collection ofinformation, such as diagnostically useful information about a celltype, tissue, organ, disease, disorder, condition, or patient.

Among certain embodiments of the present invention is the method ofdetecting a cell using a cell-targeted molecule and/or diagnosticcomposition of the invention comprising the steps of contacting a cellwith said cell-targeted molecule and/or diagnostic composition anddetecting the presence of said cell-targeted molecule and/or diagnosticcomposition. In certain embodiments, the step of contacting the cell(s)occurs in vitro. In certain embodiments, the step of contacting thecell(s) occurs in vivo. In certain embodiments, the step of detectingthe cell(s) occurs in vitro. In certain embodiments, the step ofdetecting the cell(s) occurs in vivo.

For example, a diagnostic composition of the invention may be used todetect a cell in vivo by administering to a mammalian subject acomposition comprising protein of the present invention which comprisesa detection promoting agent and then detecting the presence of theprotein of the present invention either in vitro or in vivo. Theinformation collected may regard the presence of a cell physicallycoupled with an extracellular target of the binding region of thecell-targeted molecule of the present invention and may be useful in thediagnosis, prognosis, characterization, and/or treatment of a disease,disorder, or condition. Certain compounds (e.g. polypeptides andcell-targeted molecules) of the invention, compositions (e.g.pharmaceutical compositions and diagnostic compositions) of theinvention, and methods of the invention may be used to determine if apatient belongs to a group that responds to a pharmaceutical compositionof the invention.

Certain embodiments of the polypeptides of the present invention and thecell-targeted molecules of the present invention may be utilized as animmunogen or as a component of an immunogen for the immunization and/orvaccination of a chordate.

For certain embodiments, a method of the invention is for “seeding” atissue locus within a chordate, the method comprising the step of:administering to the chordate a cell-targeted molecule of the invention,a pharmaceutical composition of the invention, or a diagnosticcomposition of the invention. In certain further embodiments, themethods of the invention for “seeding” a tissue locus are for “seeding”a tissue locus which comprises a malignant, diseased, or inflamedtissue. In certain further embodiments, the methods of the invention for“seeding” a tissue locus are for “seeding” a tissue locus whichcomprises the tissue selected from the group consisting of: diseasedtissue, tumor mass, cancerous growth, tumor, infected tissue, orabnormal cellular mass. In certain further embodiments, the methods ofthe invention for “seeding” a tissue locus comprises administering tothe chordate the cell-targeted molecule of the invention, thepharmaceutical composition of the invention, or the diagnosticcomposition of the invention comprising the heterologous T-cell epitopeselected from the group consisting of: peptides not natively presentedby the target cells of the cell-targeted molecule in MHC class Icomplexes, peptides not natively present within any protein expressed bythe target cell, peptides not natively present within the proteome ofthe target cell, peptides not natively present in the extracellularmicroenvironment of the site to be seeded, and peptides not nativelypresent in the tumor mass or infected tissue site to be targeted.

Among certain embodiments of the present invention are kits comprising acomposition of matter of the present invention, and optionally,instructions for use, additional reagent(s), and/or pharmaceuticaldelivery device(s).

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures. Theaforementioned elements of the invention may be individually combined orremoved freely in order to make other embodiments of the invention,without any statement to object to such combination or removalhereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the general arrangement of exemplary T-cell epitopepresenting effector polypeptides, including B-cell/CD4+ T-cellde-immunized variants, and cell-targeted proteins comprising the same.

FIG. 2 shows embedding T-cell epitopes into B-cell epitope regions of atoxin effector polypeptide did not significantly impair catalyticactivity. Two exemplary Diphtheria toxin-derived polypeptides comprisinga T-cell epitope embedded into a B-cell epitope region exhibited levelsof ribosome inactivation comparable to a wild-type Diphtheria toxin.

FIG. 3 shows embedding or inserting T-cell epitopes into a B-cellepitope region disrupted epitope(s) recognized by various anti-SLT-1Aantibodies by Western blot analysis.

FIG. 4 shows embedding T-cell epitopes into various B-cell epitoperegions disrupted epitope(s) recognized by various anti-SLT-1Aantibodies by Western blot analysis.

FIG. 5 shows overlays of the results of flow cytometric analysis of setsof cells receiving different treatments: untreated, treated with anexemplary cell-targeted protein of the present invention, treated withexogenous epitope-peptide and PLE, and treated with exogenousepitope-peptide only. Cells treated with three exemplary cell-targetedproteins of the present invention, each comprising a de-immunized Shigatoxin effector polypeptide comprising an embedded T-cell epitopedisrupting a B-cell epitope region, displayed the embeddedepitope-peptide complexed to MHC molecules on their cell surfaces.

DETAILED DESCRIPTION

The present invention is described more fully hereinafter usingillustrative, non-limiting embodiments, and references to theaccompanying figures. This invention may, however, be embodied in manydifferent forms and should not be construed as to be limited to theembodiments set forth below. Rather, these embodiments are provided sothat this disclosure is thorough and conveys the scope of the inventionto those skilled in the art.

In order that the present invention may be more readily understood,certain terms are defined below. Additional definitions may be foundwithin the detailed description of the invention.

As used in the specification and the appended claims, the terms “a,”“an” and “the” include both singular and the plural referents unless thecontext clearly dictates otherwise.

As used in the specification and the appended claims, the term “and/or”when referring to two species, A and B, means at least one of A and B.As used in the specification and the appended claims, the term “and/or”when referring to greater than two species, such as A, B, and C, meansat least one of A, B, or C, or at least one of any combination of A, B,or C (with each species in singular or multiple possibility).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer (or components) or group of integers (or components),but not the exclusion of any other integer (or components) or group ofintegers (or components).

Throughout this specification, the term “including” is used to mean“including but not limited to.” “Including” and “including but notlimited to” are used interchangeably.

The term “amino acid residue” or “amino acid” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.The term “polypeptide” includes any polymer of amino acids or amino acidresidues. The term “polypeptide sequence” refers to a series of aminoacids or amino acid residues which physically comprise a polypeptide. A“protein” is a macromolecule comprising one or more polypeptides orpolypeptide “chains”. A “peptide” is a small polypeptide of sizes lessthan a total of 15-20 amino acid residues. The term “amino acidsequence” refers to a series of amino acids or amino acid residues whichphysically comprise a peptide or polypeptide depending on the length.Unless otherwise indicated, polypeptide and protein sequences disclosedherein are written from left to right representing their order from anamino terminus to a carboxy terminus.

The terms “amino acid,” “amino acid residue,” “amino acid sequence,” orpolypeptide sequence include naturally occurring amino acids and, unlessotherwise limited, also include known analogs of natural amino acidsthat can function in a similar manner as naturally occurring aminoacids, such as selenocysteine, pyrrolysine, N-formylmethionine,gamma-carboxyglutamate, hydroxyprolinehypusine, pyroglutamic acid, andselenomethionine. The amino acids referred to herein are described byshorthand designations as follows in Table A:

TABLE A Amino Acid Nomenclature Name 3-letter 1-letter Alanine Ala AArginine Arg R Asparagine Asn N Aspartic Acid or Aspartate Asp DCysteine Cys C Glutamic Acid or Glutamate Glu E Glutamine Gln Q GlycineGly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys KMethionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The phrase “conservative substitution” with regard to a polypeptide,refers to a change in the amino acid composition of the polypeptide thatdoes not substantially alter the function and structure of the overallpolypeptide (see Creighton, Proteins: Structures and MolecularProperties (W. H. Freeman and Company, New York (2nd ed., 1992)).

As used herein, the term “expressed,” “expressing” or “expresses” refersto translation of a polynucleotide or nucleic acid into a polypeptide orprotein. The expressed polypeptide or proteins may remain intracellular,become a component of the cell surface membrane or be secreted into anextracellular space.

As used herein, the symbol “α” is shorthand for an immunoglobulin-typebinding region capable of binding to the biomolecule following thesymbol. The symbol “α” is used to refer to the functional characteristicof an immunoglobulin-type binding region based on its capability ofbinding to the biomolecule following the symbol.

The symbol “::” means the polypeptide regions before and after it arephysically linked together to form a continuous polypeptide.

For purposes of the present invention, the phrase “derived from” meansthat the polypeptide region comprises amino acid sequences originallyfound in a protein and which may now comprise additions, deletions,truncations, or other alterations relative to the original sequence suchthat overall function and structure are substantially conserved.

For purposes of the present invention, the term “effector” meansproviding a biological activity, such as cytotoxicity, biologicalsignaling, enzymatic catalysis, subcellular routing, and/orintermolecular binding resulting in the recruitment one or morefactor(s), and/or allosteric effects.

As used herein, the terms “subunit” and “chain” with regard tomultimeric toxins, such as, e.g., ABx toxins, are used interchangeably.

For purposes of the present invention, the phrase “CD8+ T-cellhyper-immunized” means that the molecule, when present inside anucleated, chordate cell within a living chordate, has an increasedantigenic and/or immunogenic potential regarding CD8+ T-cellantigenicity or immunogenicity. Commonly, CD8+ T-cell immunizedmolecules are capable of cellular internalization to an early endosomalcompartment of a nucleated, chordate cell due either to an inherentfeature(s) or as a component of a cell-targeted molecule.

For purposes of the present invention, the phrase “B-cell and/or CD4+T-cell de-immunized” means that the molecule has a reduced antigenicand/or immunogenic potential after administration to a mammal regardingeither B-cell antigenicity or immunogenicity and/or CD4+ T-cellantigenicity or immunogenicity.

For purposes of the present invention, the term “proteasome deliveringeffector” means a molecule that provides the biological activity oflocalizing within a cell to a subcellular compartment that is competentto result in the proteasomal degradation of the proteasome deliveringeffector molecule. Generally, this proteasome delivering biologicalactivity can be determined from the initial sub-cellular location of theproteasome delivering effector molecule in an early endosomalcompartment; however, it can also be determined earlier, e.g., from anextracellular starting location which involves passage into a cell andthrough an endosomal compartment of the cell, such as, e.g. afterendocytotic entry into that cell. Alternatively, proteasome deliveringeffector biological activity may in certain embodiments not involvepassage through any endosomal compartment of a cell before theproteasome delivering effector polypeptide is internalized and reaches acompartment competent to result in its proteasomal degradation. Theability of a given molecule to provide proteasome delivering effectorfunction(s) may be assayed by the skilled worker using techniques knownin the art.

The term “heterologous” with regard to T-cell epitope or T-cell epitopepeptide component of a polypeptide of the present invention refers to anepitope or peptide sequence which did not initially occur in thepolypeptide to be modified, but which has been added to the polypeptideusing a method of the present invention, whether added via the processesof embedding, fusion, insertion, and/or amino acid substitution asdescribed herein, or by any other engineering means. The result is amodified polypeptide comprising a T-cell epitope foreign to theoriginal, unmodified polypeptide, i.e. the T-cell epitope was notpresent in the original polypeptide.

The term “endogenous” with regard to a B-cell epitope or CD4+ T-cellepitope in a polypeptide refers to an epitope already present in thepolypeptide before being modified by a method of the present invention.

As used herein, the terms “disrupted”, “disruption,” or “disrupting,”with regard to a polypeptide region or feature within a polypeptiderefers to an alteration of at least one amino acid within the region orcomposing the feature. Amino acid alterations include various mutations,such as, e.g., a deletion, inversion, insertion, or substitution whichalter the amino acid sequence of the polypeptide. Amino acid alterationsalso include chemical changes, such as, e.g., the alteration one or moreatoms in an amino acid functional group or the addition of one or moreatoms to an amino acid functional group.

The phrase “in association with” or “associated with” with regard to aT-cell epitope or T-cell epitope peptide component of a polypeptide ofthe present invention means the T-cell epitope and polypeptide arephysically linked together, whether by covalent or non-covalentlinkages, such as, e.g., embedded or inserted within the polypeptide,fused to the polypeptide, and/or chemically conjugated to thepolypeptide.

The term “associating” with regard to the claimed invention means theact of making two molecules associated with each other or in associationwith each other.

The term “embedded” and grammatical variants thereof, with regard to aT-cell epitope or T-cell epitope peptide component of a polypeptide ofthe present invention refers to the internal replacement of one or moreamino acids within a polypeptide region with different amino acids inorder to generate a new polypeptide sequence sharing the same totalnumber of amino acid residues. Thus, the term embedded does not includeany external, terminal fusion of any additional amino acid, peptide, orpolypeptide component to the starting polypeptide nor any additionalinternal insertion of any additional amino acid residues but rather onlysubstitutions for existing amino acids. The internal replacement may beaccomplished merely by amino acid residue substitution or by a series ofsubstitutions, deletions, insertions, and/or inversions. If an insertionof one or more amino acids is used, then the equivalent number ofproximal amino acids must be deleted next to the insertion to result inan embedded T-cell epitope. This is in contrast to use of the term“inserted” with regard to T-cell epitopes in the polypeptides of thepresent invention which instead refers to a polypeptide with anincreased length equivalent to the length of the inserted T-cellepitope. Insertions include the previous even if other regions of thepolypeptide not proximal to the insertion are deleted to then decreasethe total length of the final polypeptide.

The term “fused” and grammatical variants thereof, with regard to aT-cell epitope or T-cell epitope peptide component of a polypeptide ofthe present invention refers to the external addition of four, five,six, or more amino acids to either the amino-terminus or carboxyterminus of a polypeptide in order to generate a new polypeptide whichhas a greater number of amino acid residues than the original. FusedT-cell epitopes include the addition of four, five, six, or more aminoacids to either the amino-terminus or carboxy terminus even if otherregions of the polypeptide are deleted to then decrease the total lengthof the final polypeptide as long as the new polypeptide retains aneffector function of the original polypeptide, such as, e.g., proteasomedelivering effector function.

As used herein, the term “toxin effector polypeptide” means apolypeptide that comprises a toxin-derived effector region that issufficient to provide one or more biological activities present in thetoxin from which the polypeptide was derived.

As used herein, the term “T-cell epitope delivering” means that amolecule provides the biological activity of localizing within a cell toa subcellular compartment that is competent to result in the proteasomaldegradation of a T-cell epitope carrying polypeptide region. Generally,this proteasome delivering biological activity can be determined fromthe initial sub-cellular location of the T-cell epitope deliveringmolecule in an early endosomal compartment; however, it can also bedetermined earlier, e.g., from an extracellular starting location whichinvolves passage into a cell and through an endosomal compartment of thecell, such as, e.g. after endocytotic entry into that cell.Alternatively, T-cell epitope delivering activity may in certainembodiments not involve passage through any endosomal compartment of acell before the T-cell epitope delivering molecule is internalized andreaches a compartment competent to deliver a T-cell epitope to theproteasome for degradation into a T-cell epitope peptide. EffectiveT-cell epitope delivering function can be assayed by observing MHCpresentation of the delivered T-cell epitope on a cell surface of a cellin which the T-cell epitope delivering molecule has internalized.

As used herein, a toxin effector function or activity may include, interalia, promoting cellular internalization, promoting endosome escape,directing intracellular routing to a subcellular compartment, catalyticfunctions, substrate binding, inducing apoptosis of cell, causingcytostasis, and cytotoxicity.

As used herein, the retention of a toxin-derived polypeptide effectorfunction refers to a level of toxin effector functional activity, asmeasured by an appropriate quantitative assay with reproducibility,comparable to a wild-type polypeptide (WT) control. For example, variousassays know to the skilled worker may be used to measure the enzymaticactivity and/or intracellular routing of a toxin effector polypeptide.The enzymatic polypeptide effector toxin function of a polypeptide ofthe present invention is retained if its enzymatic activity iscomparable to a wild-type polypeptide (WT) in the same assay under thesame conditions.

The term “selective cytotoxicity” with regard to the cytotoxic activityof a cytotoxic protein refers to the relative levels of cytotoxicitybetween a targeted cell population and a non-targeted bystander cellpopulation, which can be expressed as a ratio of the half-maximalcytotoxic concentration (CD₅₀) for a targeted cell type over the CD₅₀for an untargeted cell type to show preferentiality of cell killing ofthe targeted cell type.

INTRODUCTION

The present invention provides methods of generating polypeptides andcell-targeted molecules which are capable of delivering T-cell epitopepeptides to the MHC class I system of a target cell for cell surfacepresentation. The present invention also provides exemplary T-cellepitope delivering polypeptides, made using the methods of theinvention, which are capable of delivering heterologous T-cell epitopesto the MHC class I system of a target cell for cell surfacepresentation. The polypeptides created using the methods of the presentinvention, e.g. T-cell epitope delivering polypeptides and CD8+ T-cellhyper-immunized polypeptides, may be utilized as components of variousmolecules and compositions, e.g. cytotoxic therapeutics, therapeuticdelivery agents, and diagnostic molecules.

In addition, the present invention provides methods of generatingvariants of polypeptides by simultaneously reducing the probability ofB-cell antigenicity and/or immunogenicity while providing at anoverlapping position within the polypeptide a heterologous T-cellepitope for increasing the probability of T-cell immunogenicity via MHCclass I presentation. The present invention also provides exemplaryB-cell epitope de-immunized, T-cell epitope delivering polypeptides madeusing the methods of the invention, which are capable of deliveringheterologous T-cell epitopes to the MHC class I system of a target cellfor cell surface presentation. The polypeptides created using themethods of the present invention, e.g. B-cell/CD4+ T-cell de-immunizedT-cell epitope delivering polypeptides, CD8+ T-cell hyper-immunized andCD4+ T-cell de-immunized polypeptides, may be utilized as components ofvarious molecules and compositions, e.g. cytotoxic therapeutics,therapeutic delivery agents, and diagnostic molecules.

I. The General Structure of a CD8+ T-Cell Hyper-Immunized Polypeptide

The present invention involves the engineering of polypeptidescomprising various proteasome delivery effector polypeptide regions tocomprise one or more heterologous T-cell epitopes; and where upondelivery of the polypeptide to an early endosomal compartment of aeukaryotic cell, the polypeptide is capable of localizing within thecell to a subcellular compartment sufficient for delivering the one ormore heterologous T-cell epitopes to the proteasome for degradation andentry into the cell's MHC class I system. While the proteasome deliveryeffector polypeptides may come from any source, in certain embodiments,the polypeptides of the invention are derived from various proteasomedelivery effector polypeptides derived from naturally occurring proteintoxins.

A. Polypeptides Engineered to Comprise One or More Heterologous. T-CellEpitopes and a Proteasome Delivery Effector Polypeptide

The present invention contemplates the use of various polypeptides as astarting point for modification into a polypeptide of the presentinvention via the embedding, fusing, and/or inserting of one or moreheterologous T-cell epitopes. These source polypeptides should exhibit,or be predicted, to exhibit a proteasome delivery effector capability.

The predominant source of peptide epitopes entering the MHC class Ipathway are peptides resulting from proteasomal degradation of cytosolicmolecules. However, ER-localized molecules, such as viral glycoproteinsand transformed cell glycoproteins, can also be displayed by the MHCclass I system by a different route. Although this alternative route toMHC class I presentation begins in the ER, the polypeptide or proteinsource of the peptide is transported to the cytosol for proteloyticprocessing by the proteasome before being transported by TAPs to thelumen of the ER for peptide loading onto MHC class I molecules. Theexact mechanism underlying this alternative route is not clear but mightinvolve an ER-associated degradation (ERAD)-type surveillance system todetect misfolded proteins, “defective ribosomal products,” andstructures mimicking the aforementioned. This ERAD-type systemtransports certain polypeptides and proteins to proteasomes in thecytosol for degradation, which can result in the production of cytosolicantigenic peptides. In addition, research on the intracellular routingof various toxins suggests that reaching either the cytosol or theendoplasmic reticulum is sufficient for delivery of a T-cell epitopeinto the MHC class I pathway.

Therefore, polypeptides and proteins known or discovered to localize toand/or direct their own intracellular transport to the cytosol and/orendoplasmic reticulum represent a class of molecules predicted tocomprise one or more proteasome delivery effector polypeptide regionswhich exhibit a proteasome delivery function(s). Certain proteins andpolypeptides, such as, e.g., certain toxins, exhibit the ability toescape from endosomal compartments into the cytosol, thereby avoidinglysosomal degradation. Thus, polypeptides and proteins known ordiscovered to escape endosomal compartments and reach the cytosol areincluded in the class of molecules mentioned above. The exact route thepolypeptide or protein takes to the cytosol or ER is irrelevant as longas the polypeptide or protein eventually reaches a subcellular locationthat permits access to the proteasome.

In addition, certain molecules are able to reach the proteasome of acell after being localized to a lysosome. For example, foreign proteinsintroduced directly into the cytosol of a cell, such as listeriolysinand other proteins secreted by Listeria monocytogenes, can enter the MHCclass I pathway and be presented in MHC class I complexes forrecognition by effector T-cells (Villanueva M et al., J Immunol 155:5227-33 (1995)). In addition, lysosomal proteolysis, includingphagolysosome proteolysis, can produce antigenic peptides that aretranslocated into the cytosol and enter the MHC class I pathway for cellsurface presentation in a process called cross-presentation, which mayhave evolved from a canonical ERAD system (Gagnon E et al., Cell 110:119-31 (2002)). Thus, certain polypeptides and proteins known ordiscovered to localize to lysosomes may be suitable sources forpolypeptides with proteasome delivery effector regions which exhibit aproteasome delivery function(s).

The ability of a proteinaceous molecule to intracellularly route to thecytosol, ER, and/or lysosomal compartments of a cell from the startingposition of an early endosomal compartment can be determined by theskilled worker using assays known in the art. Then, the proteasomedelivery effector polypeptide regions of a source polypeptide orprotein, such as, e.g., a toxin, can be mapped and isolated by theskilled worker using standard techniques known in the art.

1. Proteasome Delivery Effector Polypeptides Derived from Toxins

The present invention contemplates the use of various polypeptidesderived from toxins as proteasome delivering effector regions. Manytoxins represent optimal sources of proteasome delivering effectorpolypeptides because of the wealth of knowledge about theirintracellular routing behaviors.

Many naturally occurring proteinaceous toxins have highly evolvedstructures optimized for directing intracellular routing in vertebratehost cells, including via endosomal escape and retrograde transportpathways.

Numerous toxins exhibit endosome escape properties, commonly via poreformation (Mandal M et al., Biochim Biophys Acta 1563: 7-17 (2002)). Forexample, diphtheria toxin and plant type II ribosome inactivatingproteins like ricin can escape from endosomes (Murphy S et al., BiochimBiophys Acta 1824: 34-43 (2006); Slominska-Wojewodzka M, Sandvig K,Antibodies 2: 236-269 (2013); Walsh M et al., Virulence 4: 774-84(2013)). Escape from endosomal compartments, including lysosomes, can bemeasured directly and quantitated using assays known in the art, suchas, e.g., using reporter assays with horseradish peroxidase, bovineserum albumin, fluorophores like Alexa 488, and toxin derivedpolypetides (see e.g. Bartz R et al., Biochem J 435: 475-87 (2011);Gilabert-Oriol R et al., Toxins 6: 1644-66 (2014)).

Many toxins direct their own intracellular routing in vertebrate hostcells. For example, the intoxication pathways of many toxins can bedescribed as a multi-step process involving 1) cellular internalizationof the toxin into host cells, 2) intracellular routing of the toxin viaone or more sub-cellular compartments, and 3) subsequent localization ofa catalytic portion of the toxin to the cytosol where host factorsubstrates are enzymatically modified. For example, this processdescribes the intoxication pathway of anthrax lethal factors, choleratoxins, diphtheria toxins, pertussis toxins, Pseudomonas exotoxins, andtype II ribosome inactivating proteins like ricin and Shiga toxins.

Similarly, recombinant toxins, modified toxin structures, and engineeredpolypeptides derived from toxins can preserve these same properties. Forexample, engineered recombinant polypeptides derived from diphtheriatoxin (DT), anthrax lethal factor (LF) toxin, and Pseudomonas exotoxin A(PE) have been used as delivery vehicles for moving polypeptides from anextracellular space to the cytosol. Any protein toxin with the intrinsicability to intracellularly route from an early endosomal compartment toeither the cytosol or the ER represents a source for a proteasomedelivery effector polypeptide which may be exploited for the purposes ofthe present invention, such as a starting component for modification oras a source for mapping a smaller proteasome delivery effector regiontherein.

For many toxins targeting eukaryotic cells, toxicity is the result of anenzymatic mechanism involving a substrate(s) in the cytosol (see TableI). These toxins have evolved toxin structures with the ability todeliver enzymatically active polypeptide regions of their holotoxins tothe cytosol. The enzymatic regions of these toxins may be used asstarting components for creating the polypeptides of the presentinvention.

TABLE I Exemplary Protein Toxin Sources of Proteasome DeliveringEffector Polypeptides Protein Toxin Substrate-Subcellular LocationAbrins sarcin-ricin loop-cytosol Anthrax lethal factor MAPKK-cytosolAspf1 sarcin-ricin loop-cytosol Bouganin sarcin-ricin loop-cytosolBryodins sarcin-ricin loop-cytosol Cholix toxin heterotrimeric Gprotein-cytosol Cinnamomin sarcin-ricin loop-cytosol Claudinsarcin-ricin loop-cytosol Clavin sarcin-ricin loop-cytosol C. difficileTcdA Ras GTPases-cytosol C. difficile TcdA Rho GTPases-cytosol C.perfringens iota Rho GTPases-cytosol cytolethal distending DNA-nucleusDianthins sarcin-ricin loop-cytosol Diphtheria toxins elongationfactor-2 (EF2)-cytosol Ebulins sarcin-ricin loop-cytosol Geloninsarcin-ricin loop-cytosol Gigantin sarcin-ricin loop-cytosol heat-labileenterotoxins heterotrimeric G protein-cytosol Maize RIPs sarcin-ricinloop-cytosol Mitogillin sarcin-ricin loop-cytosol Nigrins sarcin-ricinloop-cytosol Pertussis toxins heterotrimeric G protein-cytosol PD-Lssarcin-ricin loop-cytosol PAPs sarcin-ricin loop-cytosol Pseudomonastoxins elongation factor-2 (EF2)-cytosol Pulchellin sarcin-ricinloop-cytosol Restrictocin sarcin-ricin loop-cytosol Ricins sarcin-ricinloop-cytosol Saporins sarcin-ricin loop-cytosol Sarcins sarcin-ricinloop-cytosol Shiga toxins sarcin-ricin loop-cytosol Subtilase cytotoxinsendoplasmic chaperon-ER Trichosanthins sarcin-ricin loop-cytosol

The toxins in two toxin superfamilies, with overlapping members, arevery amenable for use in the present invention: ABx toxins andribotoxins.

ABx toxins are capable of entering eukaryotic cells and routing to thecytosol to attack their molecular targets. Similarly, ribotoxins arecapable of entering eukaryotic cells and routing to the cytosol toinactivate ribosomes. Members of both the Abx toxin and ribotoxinsuperfamilies are appropriate sources for identifying toxin-derivedpolypeptides and proteasome delivery effector polypeptides for use inthe present invention

ABx toxins, which are also referred to as binary toxins, are found inbacteria, fungi, and plants. The ABx toxins form a superfamily of toxinsthat share the structural organization of two or more polypeptide chainswith distinct functions, referred to as A and B subunits. The xrepresents the number of B subunits in the holotoxins of the members ofthe ABx family, such as, e.g., AB₁ for diphtheria toxin and AB₅ forShiga toxin. The AB5 toxin superfamily is comprised of 4 main families:cholix toxins (Ct or Ctx), pertussis toxins (Ptx), Shiga toxins (Stx),and Subtilase cytotoxins (SubAB). The cytotoxic mechanisms of AB5 toxinsinvolves subcellular routing of their A subunits within an intoxicated,eukaryotic, host cell to either the cytosol or the ER where thecatalytic A subunits act upon their enzymatic substrates representingvarious host cell proteins (see Table I).

Diphtheria toxins disrupt proteins synthesis via the catalyticADP-ribosylation of the eukaryotic elongation factor-2 (EF2). Diphtheriatoxins consists of a catalytic A subunit and a B subunit, which containsa phospholipid bilayer translocation effector domain and acell-targeting binding domain. During the diphtheria toxin intoxicationprocess, diphtheria toxins can intracellularly route their catalyticdomains to the cytosol of a eukaryotic cell, perhaps via endosomalescape (Murphy J, Toxins (Basel) 3: 294-308 (2011)). This endosomalescape mechanism may be shared with other toxins such as, e.g., anthraxlethal and edema factors, and the general ability of endosome escape isexhibited by many diverse toxins, including, e.g., certain C. difficiletoxins, gelonin, lysteriolysin, PE, ricin, and saporin (see e.g.Varkouhi A et al., J Control Release 151: 220-8 (2010); Murphy J, Toxins(Basel) 3: 294-308 (2011)).

In particular, toxins which inactivate ribosomes in the cytosol areuseful for identifying proteasome delivery effector polypeptides for usein the present invention. These toxins comprise polypeptide regionswhich simultaneously provide both cytosol targeting effector function(s)and cytotoxic ribotoxic toxin effector function(s).

With regard to the claimed invention, the phrase “ribotoxic toxineffector polypeptide” refers to a polypeptide derived from proteins,including naturally occurring ribotoxins and synthetic ribotoxins, whichis capable of effectuating ribosome inactivation in vitro, proteinsynthesis inhibition in vitro and/or in vivo, cytotoxicity, and/orcytostasis. Commonly, ribotoxic toxin effector polypeptides are derivedfrom naturally occurring protein toxins or toxin-like structures whichare altered or engineered by human intervention. However, otherpolypeptides, such as, e.g., naturally occurring enzymatic domains notnatively present in a toxin or synthetic polypeptide, are within thescope of that term as used herein (see e.g. Newton D et al., Blood 97:528-35 (2001); De Lorenzo C et al., FEBS Lett 581: 296-300 (2007); DeLorenzo C, D'Alessio G, Curr Pharm Biotechnol 9: 210-4 (2008); Menzel Cet al., Blood 111: 3830-7 (2008)). Thus, ribotoxic toxin effectorpolypeptides may be derived from synthetic or engineered proteinconstructs with increased or decreased ribotoxicity, and/or naturallyoccurring proteins that have been otherwise altered to have a non-nativecharacteristic.

The ribotoxic toxin effector polypeptides may be derived from ribotoxicdomains of proteins from diverse phyla, such as, e.g., algae, bacteria,fungi, plants, and animals. For example, polypeptides derived fromvarious ribotoxins have been linked or fused to immunoglobulin domainsor receptor ligands through chemical conjugation or recombinant proteinengineering with the hope of creating cell-type-specific cytotoxictherapeutics (Pastan I et al., Annu Rev Biochem 61: 331-54 (1992); FossF et al., Curr Top Microbiol Immunol 234: 63-81 (1998); Olsnes S,Toxicon 44: 361-70 (2004); Pastan I, et al., Nat Rev Cancer 6: 559-65(2006); Lacadena J et al., FEMS Microbiol Rev 31: 212-37 (2007); deVirgilio M et al., Toxins 2: 2699-737 (2011); Walsh M, Virulence 4:774-84 (2013); Weidle U et al., Cancer Genomics Proteomics 11: 25-38(2014)).

Ribotoxic toxin effector polypeptides may be derived from the catalyticdomains of members of the Ribosome Inactivating Protein (RIP)Superfamily of protein ribotoxins (de Virgilio M et al., Toxins 2:2699-737 (2011); Lapadula W et al., PLoS ONE 8: e72825 (2013); Walsh M,Virulence 4: 774-84 (2013)). RIPs are ribotoxic proteins expressed inalgae, bacteria, fungi, and plants which are often potent inhibitors ofeukaryotic and prokaryotic protein synthesis at sub-stoichiometricconcentrations (see Stirpe, F, Biochem J 202: 279-80 (1982)). VariousRIPs are considered promising sources for toxin effector polypeptidesequences for use in therapeutics for treating cancers (see Pastan I, etal., Nat Rev Cancer 6: 559-65 (2006); Fracasso G et al.,Ribosome-inactivating protein-containing conjugates for therapeutic use,Toxic Plant Proteins 18, pp. 225-63 (Eds. Lord J, Hartley, M. Berlin,Heidelberg: Springer-Verlag, 2010); de Virgilio M et al., Toxins 2:2699-737 (2011); Puri M et al., Drug Discov Today 17: 774-83 (2012);Walsh M, Virulence 4: 774-84 (2013)).

The most commonly used ribotoxins in recombinant cytotoxic polypeptidesinclude diphtheria toxin, Pseudomonas exotoxin A, ricin, α-sarcin,saporin, and gelonin (see Shapira A, Benhar I, Toxins 2: 2519-83 (2010);Yu C et al., Cancer Res 69: 8987-95 (2009); Fuenmayor J, Montaño R,Cancers 3: 3370-93 (2011); Weldon, FEBS J 278: 4683-700 (2011);Carreras-Sangrá N et al., Protein Eng Des Sel 25: 425-35 (2012); Lyu Mat al., Methods Enzymol 502: 167-214 (2012); Antignani, Toxins 5:1486-502 (2013); Lin H et al., Anticancer Agents Med Chem 13: 1259-66(2013); Polito L et al., Toxins 5: 1698-722 (2013); Walsh M, Virulence4: 774-84 (2013)). These ribotoxins are generally classified as ribosomeinactivating proteins (RIPs) and share a general cytotoxic mechanism ofinactivating eukaryotic ribosomes by attacking the sarcin-ricin loop(SRL) or proteins required for ribosome function which bind to the SRL.

The SRL structure is highly conserved between the three phylogeneticgroups, Archea, Bacteria and Eukarya, such that both prokaryotic andeukaryotic ribosomes share a SRL ribosomal structure (Gutell R et al.,Nucleic Acids Res 21: 3055-74 (1993); Szewczak A, Moore P, J Mol Biol247: 81-98 (1995); Glück A, Wool I, J Mol Biol 256: 838-48 (1996);Seggerson K, Moore P, RNA 4: 1203-15 (1998); Correll C et al., J MolBiol 292: 275-87 (1999)). The SRL of various species from diverse phylacan be superimposed onto a crystal structure electron density map withhigh precision (Ban N et al., Science 11: 905-20 (2000); Gabashvili I etal., Cell 100: 537-49 (2000)). The SRL is the largest universallyconserved ribosomal sequence which forms a conserved secondary structurevital to the ribosome function of translocation via the cooperation ofelongation factors, such as EF-Tu, EF-G, EF1, and EF2 (Voorhees R etal., Science 330: 835-8 (2010); Shi X et al., J Mol Biol 419: 125-38(2012); Chen K et al., PLoS One 8: e66446 (2013)). The SRL (sarcin-ricinloop) was named for being the shared target of the fungal ribotoxinsarcin and the plant type II RIP ricin.

The RIP Superfamily includes RIPs, fungal ribotoxins, and bacterialribotoxins that interfere with ribosome translocation functions (seeTable B; Brigotti M et al., Biochem J 257: 723-7 (1989)). Most RIPs,like abrin, gelonin, ricin, and saporin, irreversibly depurinate aspecific adenine in the universally conserved sarcin/ricin loop (SRL) ofthe large rRNAs of ribosomes (e.g. A4324 in animals, A3027 in fungi, andA2660 in prokaryotes). Most fungal ribotoxins, like α-sarcin,irreversibly cleave a specific bond in the SRL (e.g. the bond betweenG4325 and A4326 in animals, G3028 and A3029 in fungi, and G2661 andA2662 in prokaryotes) to catalytically inhibit protein synthesis bydamaging ribosomes (Martinez-Ruiz A et al., Toxicon 37: 1549-63 (1999);Lacadena J et al., FEMS Microbiol Rev 31: 212-37 (2007); Tan Q et al., JBiotechnol 139: 156-62 (2009)). The bacterial protein ribotoxins Ct, DT,and PE are classified in the RIP Superfamily because they can inhibitprotein synthesis by catalytically damaging ribosome function and induceapoptosis efficiently with only a few toxin molecules.

RIPs are defined by one common feature, the ability to inhibittranslation in vitro by damaging the ribosome via ribosomal RNA(rRNA)N-glycosidase activity. By 2013, over one hundred RIPs had beendescribed (Walsh M, Virulence 4: 774-84 (2013)). Most RIPs depurinate aspecific adenine residue in the universally conserved sarcin/ricin loop(SRL) of the large rRNA of both eukaryotic and prokaryotic ribosomes.The highest number of RIPs has been found in the following families:Caryophyllaceae, Sambucaceae, Cucurbitaceae, Euphorbiaceae,Phytolaccaceae, and Poaceae.

Members of the RIP family are categorized into at least three classesbased on their structures. Type I RIPs, e.g. gelonin, luffins, PAP,saporins and trichosanthins, are monomeric proteins comprising anenzymatic domain and lacking an associated targeting domain. Type IIRIPs, e.g. abrin, ricin, Shiga toxins, are multi-subunit, heteromericproteins with an enzymatic A subunit and a targeting B subunit(s)typical of binary ABx toxins (Ho M, et al., Proc Natl Acad Sci USA 106:20276-81 (2009)). Type III RIPs, e.g. barley JIP60 RIP and maize b-32RIP, are synthesized as proenzymes that require extensive proteolyticprocessing for activation (Peumans W et al., FASEB J 15: 1493-1506(2001); Mak A et al., Nucleic Acids Res 35: 6259-67 (2007)).

Although there is low sequence homology (<50% identity) between membersof the RIP family, their catalytic domains share conserved tertiarystructures which are superimposable such that key residues involved inthe depurination of the ribosome are identifiable (de Virgilio M et al.,Toxins 2: 2699-737 (2011); Walsh M, Virulence 4: 774-84 (2013)). Forexample, the catalytic domains of ricin and Shiga toxin aresuperimposable using crystallographic data despite the 18% sequenceidentity of their A-chain subunits (Fraser M et al., Nat Struct Biol 1:59-64 (1994)).

Many enzymes and polypeptide effector regions have been used to createcytotoxic components of immunotoxins such as, e.g., gelonin, saporin,pokeweed antiviral protein (PAP), bryodin, bouganin, momordin, dianthin,momorcochin, trichokirin, luffin, restrictocin, mitogillin,alpha-sarcin, Onconase®, pancreatic ribonuclease, Bax,eosinophil-derived neurotoxin, and angiogenin. In particular, potentlycytotoxic immunotoxins have been generated using polypeptides derivedfrom the RIPs: ricin, gelonin, saporin, momordin, and PAPs (PasqualucciL et al., Haematologica 80: 546-56 (1995)).

During their respective intoxication processes, cholera toxins, ricins,and Shiga toxins all subcellularly route to the ER where their catalyticdomains are then released and translocated to the cytosol. These toxinsmay take advantage of the host cell's unfolded protein machinery andERAD system to signal the host cell to export their catalytic domainsinto the cytosol (see Spooner R, Lord J, Curr Top Microbiol Immunol 357:190-40 (2012)).

The ability of a given molecule to intracellularly route to specificsub-cellular compartments may be assayed by the skilled worker usingtechniques known in the art. This includes common techniques in the artthat can localize a molecule of interest to any one of the followingsub-cellular compartments: cytosol, ER, and lysosome.

With regard to the claimed invention, the phrase “cytosol targetingtoxin effector polypeptide” refers to a polypeptide derived fromproteins, including naturally occurring ribotoxins and syntheticribotoxins, which are capable of routing intracellularly to the cytosolafter cellular internalization. Commonly, cytosolic targeting toxineffector regions are derived from naturally occurring protein toxins ortoxin-like structures which are altered or engineered by humanintervention, however, other polypeptides, such as, e.g., computationaldesigned polypeptides, are within the scope of the term as used herein(see e.g. Newton D et al., Blood 97: 528-35 (2001); De Lorenzo C et al.,FEBS Lett 581: 296-300 (2007); De Lorenzo C, D'Alessio G, Curr PharmBiotechnol 9: 210-4 (2008); Menzel C et al., Blood 111: 3830-7 (2008)).Thus, cytosolic targeting toxin effector regions may be derived fromsynthetic or engineered protein constructs with increased or decreasedribotoxicity, and/or naturally occurring proteins that have beenotherwise altered to have a non-native characteristic. The ability of agiven molecule to provide cytosol targeting toxin effector function(s)may be assayed by the skilled worker using techniques known in the art.

The cytosolic targeting toxin effector regions of the present inventionmay be derived from ribotoxic toxin effector polypeptides and oftenoverlap or completely comprise a ribotoxic toxin effector polypeptide.

2. Proteasome Delivery Effector Polypeptides Derived from OtherPolypeptide Regions or Non-Proteinaceous Materials

There are numerous proteinaceous molecules, other than toxin-derivedmolecules, which have the intrinsic ability to localize within a celland/or direct their own intracellular routing, to the cytosol, ER, orany other subcellular compartment suitable for delivery to a proteasome.Any of these polypeptides may be used directly or derivatized intoproteasome delivery effector polypeptides for use in the presentinvention as long as the intrinsic subcellular localization effectorfunction is preserved.

For example, numerous molecules are known to be able to escape fromendosomal compartments after being endocytosed into a cell, includingnumerous naturally occurring proteins and polypeptides, via numerousmechanisms, including pore formation, lipid bilayer fusion, and protonsponge effects (see e.g. Varkouhi A et al., J Control Release 151: 220-8(2010)). Non-limiting examples of non-toxin derived molecules withendosomal escape functions include: viral agents like hemagglutinin HA2;vertebrate derived polypeptides and peptides like human calcitoninderived peptides, bovine prion protein, and sweet arrow peptide;synthetic biomimetic peptides; and polymers with endosome disruptingabilities (see e.g. Varkouhi A et al., J Control Release 151: 220-8(2010)). Escape from endosomal compartments, including lysosomes, can bemeasured directly and quantitated using assays known in the art, suchas, e.g., using reporter assays with horseradish peroxidase, bovineserum albumin, fluorophores like Alexa 488, and toxin derivedpolypetides (see e.g. Bartz R et al., Biochem J 435: 475-87 (2011);Gilabert-Oriol, R et al., Toxins 6: 1644-66 (2014)).

Other examples are molecules which localize to specific intracellularcompartments. Most polypeptides comprising an endoplasmicretention/retrieval signal motif (e.g. KDEL) can localize to the ER of aeukaryotic cell from different compartments within the cell.

The ability of a polypeptide to intracellularly route to the cytosol,ER, and/or lysosomal compartments of a cell from the starting positionof an early endosomal compartment can be determined by the skilledworker using assays known in the art. Then, the proteasome deliveryeffector polypeptide regions of a source polypeptide or protein, suchas, e.g., a toxin, can be mapped and isolated by the skilled workerusing standard techniques known in the art.

3. Polypeptides Engineered to Comprise One or More Heterologous, T-CellEpitopes and a Proteasome Delivery Effector Polypeptide

Once a proteasome delivery effector polypeptide is obtained, it can beengineered into a T-cell hyper-immunized and/or B-cell/CD4+ T-cellde-immunized polypeptide of the present invention using the methods ofthe present invention. Using the methods of the present invention, oneor more T-cell epitopes are embedded, fused, or inserted into anyproteasome delivery effector polypeptide, such as, e.g., a toxineffector polypeptide which routes to the cytosol (which may include aribotoxic toxin effector polypeptide), in order to create polypeptidesof the present invention, which starting from an early endosomalcompartment are capable of delivering a T-cell epitope to the proteasomefor entry into the MHC class I pathway and subsequent MHC class Ipresentation.

A given molecule's ability to deliver T-cell epitopes to the proteasomefor entry into the MHC class I pathway of a cell may be assayed by theskilled worker using the methods described herein and/or techniquesknown in the art (see Examples, infra). Similarly, a given molecule'sability to deliver a T-cell epitope from an early endosome compartmentto a proteasome may be assayed by the skilled worker using the methodsdescribed herein and/or techniques known in the art.

A given molecule's ability to deliver a T-cell epitope from an earlyendosome compartment to a MHC class I molecule for presentation on thesurface of a cell may be assayed by the skilled worker using the methodsdescribed herein and/or techniques known in the art (see Examples,infra). Similarly, a given molecule's ability to deliver a T-cellepitope from an early endosome compartment to a MHC class I molecule maybe assayed by the skilled worker using the methods described hereinand/or techniques known in the art.

The proteasome delivery effector polypeptides modified using the methodsof the present invention are not required to be capable of inducing orpromoting cellular internalization either before or after modificationby the methods of the present invention. In order to make cell-targetedmolecules of the present invention, the polypeptides of the presentinvention may be linked, using standard techniques known in the art,with other components known to the skilled worker in order to providecell-targeting and/or cellular internalization function(s) as needed.

B. Heterologous T-Cell Epitopes

The polypeptides and cell-targeted molecules of the present inventioneach comprise one or more heterologous T-cell epitopes. A T-cell epitopeis a molecular structure which is comprised by an antigen and can berepresented by a peptide or linear amino acid sequence and. Aheterologous T-cell epitope is an epitope not already present in thesource polypeptide or starting proteasome delivery effector polypeptidethat is modified using a method of the present invention in order tocreate a T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptide of the present invention.

The heterologous T-cell epitope peptide may be incorporated into thesource polypeptide via numerous methods known to the skilled worker,including, e.g., the processes of creating one or more amino acidsubstitutions within the source polypeptide, fusing one or more aminoacids to the source polypeptide, inserting one or more amino acids intothe source polypeptide, linking a peptide to the source polypeptide,and/or a combination of the aforementioned processes. The result is amodified variant of the source polypeptide which comprises one or moreheterologous T-cell epitopes.

Although any T-cell epitope is contemplated as being used as aheterologous T-cell epitope of the present invention, certain epitopesmay be selected based on desirable properties. One objective is tocreate CD8+ T-cell hyper-immunized polypeptides, meaning that theheterologous T-cell epitope is highly immunogenic and can elicit robustimmune responses in vivo when displayed complexed with a MHC class Imolecule on the surface of a cell. In certain embodiments of thepolypeptides of the present invention, the one or more heterologousT-cell epitopes are CD8+ T-cell epitopes.

T-cell epitopes may be derived from a number of sources, includingpeptide components of proteins and peptides derived from proteinsalready known or shown to be capable of eliciting a mammalian immuneresponse. T-cell epitopes may be created or derived from variousnaturally occurring proteins. T-cell epitopes may be derived fromvarious naturally occurring proteins foreign to mammals, such as, e.g.,proteins of microorganisms. In particular, infectious microorganisms maycontain numerous proteins with known antigenic and/or immunogenicproperties or sub-regions or epitopes. T-cell epitopes may be derivedfrom mutated human proteins and/or human proteins aberrantly expressedby malignant human cells.

T-cell epitopes may be chosen or derived from a number of sourcemolecules already known to be capable of eliciting a mammalian immuneresponse, including peptides, peptide components of proteins, andpeptides derived from proteins. For example, the proteins ofintracellular pathogens with mammalian hosts are sources for T-cellepitopes. There are numerous intracellular pathogens, such as viruses,bacteria, fungi, and single-cell eukaryotes, with well-studied antigenicproteins or peptides. T-cell epitopes can be selected or identified fromhuman viruses or other intracellular pathogens, such as, e.g., bacterialike mycobacterium, fungi like toxoplasmae, and protists liketrypanosomes.

For example, there are many known immunogenic viral peptide componentsof viral proteins from human viruses. Numerous human T-cell epitopeshave been mapped to peptides within proteins from influenza A viruses,such as peptides in the proteins HA glycoproteins FE17, S139/1, C_(H)65,C05, hemagglutin 1 (HA1), hemagglutinin 2 (HA2), nonstructural protein 1and 2 (NS1 and NS 2), matrix protein 1 and 2 (M1 and M2), nucleoprotein(NP), neuraminidase (NA)), and many of these peptides have been shown toelicit human immune responses, such as by using ex vivo assay (see e.g.Assarsson E et al, J Virol 82: 12241-51 (2008); Alexander J et al., HumImmunol 71: 468-74 (2010); Wang M et al., PLoS One 5: e10533 (2010); WuJ et al., Clin Infect Dis 51: 1184-91 (2010); Tan P et al., Human Vaccin7: 402-9 (2011); Grant E et al., Immunol Cell Biol 91: 184-94 (2013);Terajima M et al., Virol J 10: 244 (2013)). Similarly, numerous humanT-cell epitopes have been mapped to peptide components of proteins fromhuman cytomegaloviruses (HCMV), such as peptides in the proteins pp65(UL83), UL128-131, immediate-early 1 (IE-1; UL123), glycoprotein B,tegument proteins, and many of these peptides have been shown to elicithuman immune responses, such as by using ex vivo assays (Schoppel K etal., J Infect Dis 175: 533-44 (1997); Elkington R et al, J Virol 77:5226-40 (2003); Gibson L et al., J Immunol 172: 2256-64 (2004); RyckmanB et al., J Virol 82: 60-70 (2008); Sacre K et al., J Virol 82: 10143-52(2008)).

While any T-cell epitope may be used in the compositions and methods ofthe present invention, certain T-cell epitopes may be preferred based ontheir known and/or empirically determined characteristics.

In many species, the MHC gene encodes multiple MHC-I molecular variants.Because MHC class I protein polymorphisms can affect antigen-MHC class Icomplex recognition by CD8+ T-cells, heterologous T-cell epitopes may bechosen using based on knowledge about certain MHC class I polymorphismsand/or the ability of certain antigen-MHC class I complexes to berecognized by T-cells of different genotypes.

There are well-defined peptide-epitopes that are known to beimmunogenic, MHC class I restricted, and/or matched with a specifichuman leukocyte antigen (HLA) variant(s). For applications in humans orinvolving human target cells, HLA-Class I-restricted epitopes can beselected or identified by the skilled worker using standard techniquesknown in the art. The ability of peptides to bind to human MHC Class Imolecules can be used to predict the immunogenic potential of putativeT-cell epitopes. The ability of peptides to bind to human MHC class Imolecules can be scored using software tools. T-cell epitopes may bechosen for use as a heterologous T-cell epitope component of the presentinvention based on the peptide selectivity of the HLA variants encodedby the alleles more prevalent in certain human populations. For example,the human population is polymorphic for the alpha chain of MHC class Imolecules, and the variable alleles are encoded by the HLA genes.Certain T-cell epitopes may be more efficiently presented by a specificHLA molecule, such as, e.g., the commonly occurring HLA variants encodedby the HLA-A allele groups HLA-A2 and HLA-A3.

When choosing T-cell epitopes for use as a heterologous T-cell epitopecomponent of the present invention, multiple factors in the process ofepitope selection by MHC class I molecules may be considered that caninfluence epitope generation and transport to receptive MHC class Imolecules, such as, e.g., the epitope specificity of the followingfactors in the target cell: proteasome, ERAAP/ERAP1, tapasin, and TAPscan (see e.g. Akram A, Inman R, Clin Immunol 143: 99-115 (2012)).

When choosing T-cell epitopes for use as a heterologous T-cell epitopecomponent of the present invention, epitope-peptides may be selectedwhich best match the MHC Class I molecules present in the cell-type orcell populations to be targeted. Different MHC class I molecules exhibitpreferential binding to particular peptide sequences, and particularpeptide-MHC class I variant complexes are specifically recognized by theTCRs of effector T-cells. The skilled worker can use knowledge about MHCclass I molecule specificities and TCR specificities to optimize theselection of heterologous T-cell epitopes used in the present invention.

In addition, multiple immunogenic T-cell epitopes for MHC class Ipresentation may be embedded in the same polypeptide component(s) foruse in the targeted delivery of a plurality of T-cell epitopessimultaneously.

C. Proteasome Delivery Effector Polypeptides which Comprise One or MoreHeterologous T-Cell Epitopes Embedded or Inserted to Disrupt anEndogenous B-Cell and/or CD4+ T-Cell Epitope Region

Despite the attractiveness of using proteasome delivery effectorpolypeptides as components of therapeutics, many polypeptides areimmunogenic in extracellular spaces when administered to vertebrates.Unwanted immunogenicity in protein therapeutics has resulted in reducedefficacy, unpredictable pharmacokinetics, and undesirable immuneresponses that limit dosages and repeat administrations. In efforts tode-immunize therapeutics, one main challenge is silencing or disruptingimmunogenic epitopes within a polypeptide effector domain, e.g. itscytosolic targeting domain, while retaining the desired polypeptideeffector function(s), such as, e.g., proteasome delivery. In addition,it is a significant challenge to disrupt immune epitopes by amino acidsubstitution in a polypeptide structure while preserving its functionwhile simultaneously adding one or more T-cell epitopes that will not berecognized by the immune system until after cellular internalization,processing, and cell-surface presentation by a target cell. Solving thischallenge enables the creation of polypeptides which exhibit desiredCD8+ T-cell immunogenicity while reducing undesired B-cell and CD4+T-cell immunogenicity—referred to herein as “CD8+ T-cell hyper-immunizedand/or B-cell/CD4+ T-cell de-immunized” molecules or “T-cell epitopedelivering and/or B-cell/CD4+ T-cell de-immunized” molecules.

II. The General Structure of Cell-Targeted Molecules Comprising T-CellEpitope Delivering, CD8+ T-Cell Hyper-Immunized Polypeptides of theInvention

The polypeptides of the present invention may be coupled to numerousother polypeptides, agents, and moieties to create cell-targetedmolecules, such as, e.g. cytotoxic, cell-targeted proteins of thepresent invention. Cytotoxic polypeptides and proteins may beconstructed using the T-cell epitope comprising proteasome deliveringeffector polypeptides of the invention and the addition ofcell-targeting components, such as, e.g., a binding region capable ofexhibiting high affinity binding to an extracellular target biomoleculephysically-coupled to the surface of a specific cell type(s). Inaddition, the B-cell epitope de-immunized polypeptides of the presentinvention, whether toxic or non-toxic, may be used as components ofnumerous useful molecules for administration to mammals.

A. Cell-Targeted Molecules Comprising a Proteasome Effector PolypeptideComprising a Heterologous T-Cell Epitope

The present invention includes cell-targeted molecules, eachcomprising 1) a cell-targeting binding region and 2) a proteasomedelivering effector polypeptide of the invention which comprises aheterologous T-cell epitope.

Cell-Targeting Moeities

Certain molecules of the present invention comprise a T-cellhyper-immunized proteasome delivering effector polypeptide of thepresent invention linked to a cell-targeting moiety comprising a bindingregion capable of specifically binding an extracellular targetbiomolecule. In certain embodiments, the molecules of the presentinvention comprise a single polypeptide or protein such that the T-cellhyper-immunized proteasome delivering effector polypeptide andcell-targeting binding region are fused together to form a continuouspolypeptide chain or cell-targeting fusion protein.

Cell-targeting moieties of the cell-targeted molecules of the presentinvention comprise molecular structures, that when linked to apolypeptide of the present invention, are each capable of bringing thecell-targeted molecule within close proximity to specific cells based onmolecular interactions on the surfaces of those specific cells.Cell-targeting moieties include ligand and polypeptides which bind tocell-surface targets.

One type of cell-targeting moiety is a proteinaceous binding region.Binding regions of the cell-targeted molecules of the present inventioncomprise one or more polypeptides capable of selectively andspecifically binding an extracellular target biomolecule. Bindingregions may comprise one or more various polypeptide moieties, such asligands whether synthetic or naturally occurring ligands and derivativesthereof, immunoglobulin derived domains, synthetically engineeredscaffolds as alternatives to immunoglobulin domains, and the like. Theuse of proteinaceous binding regions in cell-targeting molecules of theinvention allows for the creation of cell-targeting molecules which aresingle-chain, cell-targeting proteins.

There are numerous binding regions known in the art that are useful fortargeting polypeptides to specific cell-types via their bindingcharacteristics, such as ligands, monoclonal antibodies, engineeredantibody derivatives, and engineered alternatives to antibodies.

According to one specific, but non-limiting aspect, the binding regionof the cell-targeted molecule of the present invention comprises anaturally occurring ligand or derivative thereof that retains bindingfunctionality to an extracellular target biomolecule, commonly a cellsurface receptor. For example, various cytokines, growth factors, andhormones known in the art may be used to target the cell-targetedmolecules of the present invention to the cell-surface of specific celltypes expressing a cognate cytokine receptor, growth factor receptor, orhormone receptor. Certain non-limiting examples of ligands include(alternative names are indicated in parentheses) B-cell activatingfactors (BAFFs, APRIL), colony stimulating factors (CSFs), epidermalgrowth factors (EGFs), fibroblast growth factors (FGFs), vascularendothelial growth factors (VEGFs), insulin-like growth factors (IGFs),interferons, interleukins (such as IL-2, IL-6, and IL-23), nerve growthfactors (NGFs), platelet derived growth factors, transforming growthfactors (TGFs), and tumor necrosis factors (TNFs).

According to certain other embodiments, the binding region comprises asynthetic ligand capable of binding an extracellular target biomolecule.One non-limiting example is antagonists to cytotoxic T-lymphocyteantigen 4 (CTLA-4).

According to one specific, but non-limiting aspect, the binding regionmay comprise an immunoglobulin-type binding region. The term“immunoglobulin-type binding region” as used herein refers to apolypeptide region capable of binding one or more target biomolecules,such as an antigen or epitope. Binding regions may be functionallydefined by their ability to bind to target molecules.Immunoglobulin-type binding regions are commonly derived from antibodyor antibody-like structures; however, alternative scaffolds from othersources are contemplated within the scope of the term.

Immunoglobulin (Ig) proteins have a structural domain known as an Igdomain. Ig domains range in length from about 70-110 amino acid residuesand possess a characteristic Ig-fold, in which typically 7 to 9antiparallel beta strands arrange into two beta sheets which form asandwich-like structure. The Ig fold is stabilized by hydrophobic aminoacid interactions on inner surfaces of the sandwich and highly conserveddisulfide bonds between cysteine residues in the strands. Ig domains maybe variable (IgV or V-set), constant (IgC or C-set) or intermediate (IgIor I-set). Some Ig domains may be associated with a complementaritydetermining region (CDR) which is important for the specificity ofantibodies binding to their epitopes. Ig-like domains are also found innon-immunoglobulin proteins and are classified on that basis as membersof the Ig superfamily of proteins. The HUGO Gene Nomenclature Committee(HGNC) provides a list of members of the Ig-like domain containingfamily.

An immunoglobulin-type binding region may be a polypeptide sequence ofan antibody or antigen-binding fragment thereof wherein the amino acidsequence has been varied from that of a native antibody or an Ig-likedomain of a non-immunoglobulin protein, for example by molecularengineering or selection by library screening. Because of the relevanceof recombinant DNA techniques and in vitro library screening in thegeneration of immunoglobulin-type binding regions, antibodies can beredesigned to obtain desired characteristics, such as smaller size, cellentry, or other therapeutic improvements. The possible variations aremany and may range from the changing of just one amino acid to thecomplete redesign of, for example, a variable region. Typically, changesin the variable region will be made in order to improve theantigen-binding characteristics, improve variable region stability, orreduce the potential for immunogenic responses.

There are numerous immunoglobulin-type binding regions contemplated ascomponents of the present invention. In certain embodiments, theimmunoglobulin-type binding region is derived from an immunoglobulinbinding region, such as an antibody paratope capable of binding anextracellular target biomolecule. In certain other embodiments, theimmunoglobulin-type binding region comprises an engineered polypeptidenot derived from any immunoglobulin domain but which functions like animmunoglobulin binding region by providing high-affinity binding to anextracellular target biomolecule. This engineered polypeptide mayoptionally include polypeptide scaffolds comprising or consistingessentially of complementary determining regions from immunoglobulins asdescribed herein.

There are also numerous binding regions in the prior art that are usefulfor targeting polypeptides to specific cell-types via theirhigh-affinity binding characteristics. In certain embodiments, thebinding region of the present proteins is selected from the group whichincludes single-domain antibody domains (sdAbs), nanobodies, heavy-chainantibody domains derived from camelids (V_(H)H fragments), bivalentnanobodies, heavy-chain antibody domains derived from cartilaginousfishes, immunoglobulin new antigen receptors (IgNARs), V_(NAR)fragments, single-chain variable (scFv) fragments, multimerizing scFvfragments (diabodies, triabodies, tetrabodies), bispecific tandem scFvfragments, disulfide stabilized antibody variable (Fv) fragments,disulfide stabilized antigen-binding (Fab) fragments consisting of theV_(L), V_(H), C_(L) and C_(H) 1 domains, divalent F(ab′)2 fragments, Fdfragments consisting of the heavy chain and C_(H)1 domains, single chainFv-C_(H)3 minibodies, bispecific minibodies, dimeric C_(H)2 domainfragments (C_(H)2D), Fc antigen binding domains (Fcabs), isolatedcomplementary determining region 3 (CDR3) fragments, constrainedframework region 3, CDR3, framework region 4 (FR3-CDR3-FR4)polypeptides, small modular immunopharmaceutical (SMIP) domains, and anygenetically manipulated counterparts of the foregoing that retain itsparatope and binding function (see Saerens D et al., Curr. Opin.Pharmacol 8: 600-8 (2008); Dimitrov D, MAbs 1: 26-8 (2009); Weiner L,Cell 148: 1081-4 (2012); Ahmad Z et al., Clin Dev Immunol 2012: 980250(2012)).

In accordance with certain other embodiments, the binding regionincludes engineered, alternative scaffolds to immunoglobulin domainsthat exhibit similar functional characteristics, such as high-affinityand specific binding of target biomolecules, and enables the engineeringof improved characteristics, such as greater stability or reducedimmunogenicity. For certain embodiments of the cell-targeted proteins ofthe present invention, the binding region is selected from the groupwhich includes engineered, fibronectin-derived, 10^(th) fibronectin typeIII (10Fn3) domain (monobodies, AdNectins™, or AdNexins™); engineered,tenascin-derived, tenascin type III domain (Centryns™); engineered,ankyrin repeat motif containing polypeptide (DARPins™); engineered,low-density-lipoprotein-receptor-derived, A domain (LDLR-A) (Avimers™);lipocalin (anticalins); engineered, protease inhibitor-derived, Kunitzdomain; engineered, Protein-A-derived, Z domain (Affibodies™);engineered, gamma-B crystalline-derived scaffold or engineered,ubiquitin-derived scaffold (Affilins); Sac7d-derived polypeptides(Nanoffitins® or affitins); engineered, Fyn-derived, SH2 domain(Fynomers®); miniproteins; C-type lectin-like domain scaffolds;engineered antibody mimics; and any genetically manipulated counterpartsof the foregoing that retains its binding functionality (Wörn A,Plückthun A, J Mol Biol 305: 989-1010 (2001); Xu L et al., Chem Biol 9:933-42 (2002); Wikman M et al., Protein Eng Des Sel 17: 455-62 (2004);Binz H et al., Nat Biotechnol 23: 1257-68 (2005); Hey T et al., TrendsBiotechnol 23:514-522 (2005); Holliger P, Hudson P, Nat Biotechnol 23:1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17: 653-8 (2006);Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007); Byla P et al., JBiol Chem 285: 12096 (2010); Zoller F et al., Molecules 16: 2467-85(2011)).

Any of the above binding regions may be used as a component of thepresent invention as long as the binding region component has adissociation constant of 10⁻⁵ to 10⁻¹² moles per liter, preferably lessthan 200 nanomolar (nM), towards an extracellular target biomolecule.

Certain cell-targeted molecules of the present invention comprise apolypeptide of the present invention linked to an extracellular targetbiomolecule specific binding region comprising one or more polypeptidescapable of selectively and specifically binding an extracellular targetbiomolecule. Extracellular target biomolecules may be selected based onnumerous criteria.

Extracellular Target Biomolecules of the Cell-Targeting Moieties

Certain binding regions of the cell-targeted molecules of the presentinvention comprise a polypeptide region capable of binding specificallyto an extracellular target biomolecule, preferably which isphysically-coupled to the surface of a cell type of interest, such as acancer cell, tumor cell, plasma cell, infected cell, or host cellharboring an intracellular pathogen.

The term “target biomolecule” refers to a biological molecule, commonlya protein or a protein modified by post-translational modifications,such as glycosylation, which is capable of being bound by a bindingregion to target a protein to a specific cell-type or location within anorganism. Extracellular target biomolecules may include variousepitopes, including unmodified polypeptides, polypeptides modified bythe addition of biochemical functional groups, and glycolipids (see e.g.U.S. Pat. No. 5,091,178; EP 2431743). It is desirable that anextracellular target biomolecule be endogenously internalized or bereadily forced to internalize upon interaction with a cell-targetedmolecule of the present invention.

For purposes of the present invention, the term “extracellular” withregard to modifying a target biomolecule refers to a biomolecule thathas at least a portion of its structure exposed to the extracellularenvironment. Extracellular target biomolecules include cell membranecomponents, transmembrane spanning proteins, cell membrane-anchoredbiomolecules, cell-surface-bound biomolecules, and secretedbiomolecules.

With regard to the present invention, the phrase “physically coupled”when used to describe a target biomolecule means both covalent and/ornon-covalent intermolecular interactions that couple the targetbiomolecule, or a portion thereof, to the outside of a cell, such as aplurality of non-covalent interactions between the target biomoleculeand the cell where the energy of each single interaction is on the orderof about 1-5 kiloCalories (e.g. electrostatic bonds, hydrogen bonds, Vander Walls interactions, hydrophobic forces, etc.). All integral membraneproteins can be found physically coupled to a cell membrane, as well asperipheral membrane proteins. For example, an extracellular targetbiomolecule might comprise a transmembrane spanning region, a lipidanchor, a glycolipid anchor, and/or be non-covalently associated (e.g.via non-specific hydrophobic interactions and/or lipid bindinginteractions) with a factor comprising any one of the foregoing.

The binding regions of the cell-targeted molecules of the presentinvention may be designed or selected based on numerous criteria, suchas the cell-type specific expression of their target biomolecules and/orthe physical localization of their target biomolecules with regard tospecific cell types. For example, certain cytotoxic proteins of thepresent invention comprise binding domains capable of bindingcell-surface targets which are expressed exclusively by only onecell-type to the cell surface.

All nucleated vertebrate cells are believed to be capable of presentingintracelular peptide epitopes using the MHC class I system. Thus,extracellular target biomolecules of the cell-targeted molecules of theinvention may in principle target any nucleated vertebrate cell forT-cell epitope delivery into the MHC class I presentation pathway.

Extracellular target biomolecules of the binding region of thecell-targeted molecules of the present invention may include biomarkersover-proportionately or exclusively present on cancer cells, immunecells, and cells infected with intracellular pathogens, such as viruses,bacteria, fungi, prions, or protozoans.

The skilled worker, using techniques known in the art, can link theT-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides of the present invention to various other molecules totarget specific extracellular target biomolecules physically coupled tocells and promote target cell internalization. For example, apolypeptide of the invention may be linked to cell-surface receptortargeting molecule which is more readily endocytosed, such as, e.g., viareceptor mediated endocytosis, or to a molecule which promotes cellularinternalization via mechanisms at the cell surface, such as, e.g.promoting clathrin coated pit assembly, phospholipid layer deformation,and/or tubular invagination. The ability of a cell-targeting moiety tofacilitate cellular internalization after target binding may bedetermined using assays known to the skilled worker.

Endoplasmic Reticulum Retention/Retrieval Signal Motif of a Member ofthe KDEL Family

For purposes of the present invention, the phrase “endoplasmic reticulumretention/retrieval signal motif,” KDEL-type signal motif, or signalmotif refers to any member of the KDEL family capable of functioningwithin a eukaryotic cell to promote subcellular localization of aprotein to the endoplasmic reticulum via KDEL receptors.

The carboxy-terminal lysine-asparagine-glutamate-leucine (KDEL) sequenceis a canonical, endoplasmic reticulum retention and retrieval signalmotif for soluble proteins in eukaryotic cells and is recognized by theKDEL receptors (see, Capitani M, Sallese M, FEBS Lett 583: 3863-71(2009), for review). The KDEL family of signal motifs includes manyKDEL-like motifs, such as HDEL, RDEL, WDEL, YDEL, HEEL, KEEL, REEL,KFEL, KIEL, DKEL, KKEL, HNEL, HTEL, KTEL, and HVEL, all of which arefound at the carboxy-terminals of proteins which are known to beresidents of the lumen of the endoplasmic reticulum of throughoutmultiple phylogenetic kingdoms (Munro S, Pelham H, Cell 48: 899-907(1987); Raykhel I et al., J Cell Biol 179: 1193-204 (2007)). The KDELsignal motif family includes at least 46 polypeptide variants shownusing synthetic constructs (Raykhel, J Cell Biol 179: 1193-204 (2007)).Additional KDEL signal motifs include ALEDEL, HAEDEL, HLEDEL, KLEDEL,IRSDEL, ERSTEL, and RPSTEL (Alanen H et al., J Mol Biol 409: 291-7(2011)). A generalized consensus motif representing the majority of KDELsignal motifs has been described as [KRHQSA]-[DENQ]-E-L (Hulo N et al.,Nucleic Acids Res 34: D227-30 (2006)).

Proteins containing KDEL family signal motifs are bound by KDELreceptors distributed throughout the Golgi complex and transported tothe endoplasmic reticulum by a microtubule-dependent mechanism forrelease into the lumen of the endoplasmic reticulum (Griffiths G et al.,J Cell Biol 127: 1557-74 (1994); Miesenbock G, Rothman J, J Cell Biol129: 309-19 (1995)). KDEL receptors dynamically cycle between the Golgicomplex and endoplasmic reticulum (Jackson M et al., EMBO J. 9: 3153-62(1990); Schutze M et al., EMBO J 13: 1696-1705 (1994)).

For purposes of the present invention, the members of the KDEL familyinclude synthetic signal motifs able to function within a eukaryoticcell to promote subcellular localization of a protein to the endoplasmicreticulum via KDEL receptors. In other words, some members of the KDELfamily might not occur in nature or have yet to be observed in naturebut have or may be constructed and empirically verified using methodsknown in the art; see e.g., Raykhel I et al., J Cell Biol 179: 1193-204(2007).

As a component of certain embodiments of the polypeptides andcell-targeted molecules of the present invention, the KDEL-type signalmotif is physically located, oriented, or arranged within thepolypeptide or cell-targeted protein such that it is on acarboxy-terminal.

For the purposes of the present invention, the specific order ororientation is not fixed for the T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptide and the cell-targetingbinding region in relation to each other or the entire, cell-targeted,fusion protein's N-terminal(s) and C-terminal(s) (see e.g. FIG. 1).

The general structure of the cell-targeted molecules of the presentinvention is modular, in that various, diverse cell-targeting bindingregions may be used with various CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptides to provide for diversetargeting of various extracellular target biomolecules and thustargeting of cytotoxicity, cytostasis, and/or exogenous materialdelivery to various diverse cell types. CD8+ T-cell hyper-immunized andB-cell/CD4+ T-cell de-immunized polypeptides which do not result inT-cell epitope presentation and/or are not cytotoxic due to impropersubcellular routing may still be useful as components of cell targetedmolecules for delivering exogenous materials into cells, such as, e.g.,a T-cell epitope or antigen.

III. Linkages Connecting Polypeptide Components of the Invention and/ortheir Subcomponents

Individual cell-targeting moiety, polypeptide, and/or protein componentsof the present invention, e.g. the cell-targeting binding regions andCD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides, may be suitably linked to each other via one or morelinkers well known in the art and/or described herein. Individualpolypeptide subcomponents of the binding regions, e.g. heavy chainvariable regions (V_(H)), light chain variable regions (V_(L)), CDR,and/or ABR regions, may be suitably linked to each other via one or morelinkers well known in the art and/or described herein (see e.g. WeisserN, Hall J, Biotechnol Adv 27: 502-20 (2009); Chen X et al., Adv DrugDeliv Rev 65: 1357-69 (2013)). Protein components of the invention,e.g., multi-chain binding regions, may be suitably linked to each otheror other polypeptide components of the invention via one or more linkerswell known in the art. Peptide components of the invention, e.g., KDELfamily endoplasmic reticulum retention/retrieval signal motifs, may besuitably linked to another component of the invention via one or morelinkers, such as a proteinaceous linker, which are well known in theart.

Suitable linkers are generally those which allow each polypeptidecomponent of the present invention to fold with a three-dimensionalstructure very similar to the polypeptide components producedindividually without any linker or other component. Suitable linkersinclude single amino acids, peptides, polypeptides, and linkers lackingany of the aforementioned such as various non-proteinaceous carbonchains, whether branched or cyclic (see e.g. Chen X et al., Adv DrugDeliv Rev 65: 1357-69 (2013)).

Suitable linkers may be proteinaceous and comprise one or more aminoacids, peptides, and/or polypeptides. Proteinaceous linkers are suitablefor both recombinant fusion proteins and chemically linked conjugates. Aproteinaceous linker typically has from about 2 to about 50 amino acidresidues, such as, e.g., from about 5 to about 30 or from about 6 toabout 25 amino acid residues. The length of the linker selected willdepend upon a variety of factors, such as, e.g., the desired property orproperties for which the linker is being selected (see e.g. Chen X etal., Adv Drug Deliv Rev 65: 1357-69 (2013)).

Suitable linkers may be non-proteinaceous, such as, e.g. chemicallinkers (see e.g. Dosio F et al., Toxins 3: 848-83 (2011); Feld J etal., Oncotarget 4: 397-412 (2013)). Various non-proteinaceous linkersknown in the art may be used to link cell-targeting moieties to the CD8+T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptide components, such as linkers commonly used to conjugateimmunoglobulin-derived polypeptides to heterologous polypeptides. Forexample, polypeptide regions may be linked using the functional sidechains of their amino acid residues and carbohydrate moieties such as,e.g., a carboxy, amine, sulfhydryl, carboxylic acid, carbonyl, hydroxyl,and/or cyclic ring group. For example, disulfide bonds and thioetherbonds may be used to link two or more polypeptides (see e.g. FitzgeraldD et al., Bioconjugate Chem 1: 264-8 (1990); Pasqualucci L et al.,Haematologica 80: 546-56 (1995)). In addition, non-natural amino acidresidues may be used with other functional side chains, such as ketonegroups (see e.g. Sun S et al., Chembiochem Jul. 18 (2014); Tian F etal., Proc Natl Acad Sci USA 111: 1766-71 (2014)). Examples ofnon-proteinaceous chemical linkers include but are not limited toN-succinimidyl (4-iodoacetyl)-aminobenzoate, S—(N-succinimidyl)thioacetate (SATA),N-succinimidyl-oxycarbonyl-cu-methyl-a-(2-pyridyldithio) toluene (SMPT),N-succinimidyl 4-(2-pyridyldithio)-pentanoate (SPP), succinimidyl4-(N-maleimidomethyl) cyclohexane carboxylate (SMCC or MCC),sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate,4-succinimidyl-oxycarbonyl-α-(2-pyridyldithio) toluene,sulfosuccinimidyl-6-(α-methyl-α-(pyridyldithiol)-toluamido) hexanoate,N-succinimidyl-3-(-2-pyridyldithio)-proprionate (SPDP), succinimidyl6(3(−(-2-pyridyldithio)-proprionamido) hexanoate, sulfosuccinimidyl6(3(−(-2-pyridyldithio)-propionamido) hexanoate, maleimidocaproyl (MC),maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB),3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS), alpha-alkylderivatives, sulfoNHS-ATMBA (sulfosuccinimidylN-[3-(acetylthio)-3-methylbutyryl-beta-alanine]), sulfodicholorphenol,2-iminothiolane, 3-(2-pyridyldithio)-propionyl hydrazide, Ellman'sreagent, dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine (seee.g. Thorpe P et al., Eur J Biochem 147: 197-206 (1985); Thorpe P etal., Cancer Res 47: 5924-31 (1987); Thorpe P et al., Cancer Res 48:6396-403 (1988); Grossbard M et al., Blood 79: 576-85 (1992); Lui C etal., Proc Natl Acad Sci USA 93: 8618-23 (1996); Doronina S et al., NatBiotechnol 21: 778-84 (2003); Feld J et al., Oncotarget 4: 397-412(2013)).

Suitable linkers, whether proteinaceous or non-proteinaceous, mayinclude, e.g., protease sensitive, environmental redox potentialsensitive, pH sensitive, acid cleavable, photocleavable, and/or heatsensitive linkers (see e.g. Dosio F et al., Toxins 3: 848-83 (2011);Chen X et al., Adv Drug Deliv Rev 65: 1357-69 (2013); Feld J et al.,Oncotarget 4: 397-412 (2013)).

Proteinaceous linkers may be chosen for incorporation into recombinantfusion cell-targeted molecules of the present invention. For recombinantfusion cell-targeted proteins of the invention, linkers typicallycomprise about 2 to 50 amino acid residues, preferably about 5 to 30amino acid residues (Argos P, J Mol Biol 211: 943-58 (1990); WilliamsonM, Biochem J 297: 240-60 (1994); George R, Heringa J, Protein Eng 15:871-9 (2002); Kreitman R, AAPS J 8: E532-51 (2006)). Commonly,proteinaceous linkers comprise a majority of amino acid residues withpolar, uncharged, and/or charged residues, such as, e.g., threonine,proline, glutamine, glycine, and alanine (see e.g. Huston J et al. ProcNatl Acad Sci U.S.A. 85: 5879-83 (1988); Pastan I et al., Annu Rev Med58: 221-37 (2007); Li J et al., Cell Immunol 118: 85-99 (1989); Cumber Aet al. Bioconj Chem 3: 397-401 (1992); Friedman P et al., Cancer Res 53:334-9 (1993); Whitlow M et al., Protein Engineering 6: 989-95 (1993);Siegall C et al., J Immunol 152: 2377-84 (1994); Newton et al.Biochemistry 35: 545-53 (1996); Ladurner et al. J Mol Biol 273: 330-7(1997); Kreitman R et al., Leuk Lymphoma 52: 82-6 (2011); U.S. Pat. No.4,894,443). Non-limiting examples of proteinaceous linkers includealanine-serine-glycine-glycine-proline-glutamate (ASGGPE),valine-methionine (VM), alanine-methionine (AM), AM(G_(2 to 4)S)_(x)AMwhere G is glycine, S is serine, and x is an integer from 1 to 10.

Proteinaceous linkers may be selected based upon the properties desired.Proteinaceous linkers may be chosen by the skilled worker with specificfeatures in mind, such as to optimize one or more of the fusionmolecule's folding, stability, expression, solubility, pharmacokineticproperties, pharmacodynamic properties, and/or the activity of the fuseddomains in the context of a fusion construct as compared to the activityof the same domain by itself. For example, proteinaceous linkers may beselected based on flexibility, rigidity, and/or cleavability (see e.g.Chen X et al., Adv Drug Deliv Rev 65: 1357-69 (2013)). The skilledworker may use databases and linker design software tools when choosinglinkers. Certain linkers may be chosen to optimize expression (see e.g.Turner D et al., J Immunol Methods 205: 43-54 (1997)). Certain linkersmay be chosen to promote intermolecular interactions between identicalpolypeptides or proteins to form homomultimers or different polypeptidesor proteins to form heteromultimers. For example, proteinaceous linkersmay be selected which allow for desired non-covalent interactionsbetween polypeptide components of the cell-targeted proteins of theinvention, such as, e.g., interactions related to the formation dimersand other higher order multimers (see e.g. U.S. Pat. No. 4,946,778).

Flexible proteinaceous linkers are often greater than 12 amino acidresidues long and rich in small, non-polar amino acid residues, polaramino acid residues, and/or hydrophilic amino acid residues, such as,e.g., glycines, serines, and threonines (see e.g. Bird R et al., Science242: 423-6 (1988); Friedman P et al., Cancer Res 53: 334-9 (1993);Siegall C et al., J Immunol 152: 2377-84 (1994)). Flexible proteinaceouslinkers may be chosen to increase the spatial separation betweencomponents and/or to allow for intramolecular interactions betweencomponents. For example, various “GS” linkers are known to the skilledworker and are composed of multiple glycines and/or one or more serines,sometimes in repeating units, such as, e.g., (G_(x)S)_(n), (S_(x)G)_(n),(GGGGS)_(n), and (G)_(n). in which x is 1 to 6 and n is 1 to 30 (seee.g. WO 96/06641). Non-limiting examples of flexible proteinaceouslinkers include GKSSGSGSESKS, GSTSGSGKSSEGKG, GSTSGSGKSSEGSGSTKG,GSTSGSGKPGSGEGSTKG, EGKSSGSGSESKEF, SRSSG, and SGSSC.

Rigid proteinaceous linkers are often stiff alpha-helical structures andrich in proline residues and/or one or more strategically placedprolines (see Chen X et al., Adv Drug Deliv Rev 65: 1357-69 (2013)).Rigid linkers may be chosen to prevent intramolecular interactionsbetween linked components.

Suitable linkers may be chosen to allow for in vivo separation ofcomponents, such as, e.g., due to cleavage and/or environment-specificinstability (see Dosio F et al., Toxins 3: 848-83 (2011); Chen X et al.,Adv Drug Deliv Rev 65: 1357-69 (2013)). In vivo cleavable proteinaceouslinkers are capable of unlinking by proteolytic processing and/orreducing environments often at a specific site within an organism orinside a certain cell type (see e.g. Doronina S et al., Bioconjug Chem17: 144-24 (2006); Erickson H et al., Cancer Res 66: 4426-33 (2006)). Invivo cleavable proteinaceous linkers often comprise protease sensitivemotifs and/or disulfide bonds formed by one or more cysteine pairs (seee.g. Pietersz G et al., Cancer Res 48: 4469-76 (1998); The J et al., JImmunol Methods 110: 101-9 (1998); see Chen X et al., Adv Drug Deliv Rev65: 1357-69 (2013)). In vivo cleavable proteinaceous linkers may bedesigned to be sensitive to proteases that exist only at certainlocations in an organism, compartments within a cell, and/or becomeactive only under certain physiological or pathological conditions (suchas, e.g., proteases with abnormally high levels, proteases overexpressedat certain disease sites, and proteases specifically expressed by apathogenic microorganism). For example, there are proteinaceous linkersknown in the art which are cleaved by proteases present onlyintracellularly, proteases present only within specific cell types, andproteases present only under pathological conditions like cancer orinflammation, such as, e.g., R-x-x-R motif andAMGRSGGGCAGNRVGSSLSCGGLNLQAM.

In certain embodiments of the cell-targeted molecules of the presentinvention, a linker may be used which comprises one or more proteasesensitive sites to provide for cleavage by a protease present within atarget cell. In certain embodiments of the cell-targeted molecules ofthe invention, a linker may be used which is not cleavable to reduceunwanted toxicity after administration to a vertebrate organism.

Suitable linkers may include, e.g., protease sensitive, environmentalredox potential sensitive, pH sensitive, acid cleavable, photocleavable,and/or heat sensitive linkers, whether proteinaceous ornon-proteinaceous (see Chen X et al., Adv Drug Deliv Rev 65: 1357-69(2013)).

Suitable cleavable linkers may include linkers comprising cleavablegroups which are known in the art such as, e.g., Zarling D et al., JImmunol 124: 913-20 (1980); Jung S, Moroi M, Biochem Biophys Acta 761:152-62 (1983); Bouizar Z et al., Eur J Biochem 155: 141-7 (1986); Park Let al., J Biol Chem 261: 205-10 (1986); Browning J, Ribolini A, JImmunol 143: 1859-67 (1989); Joshi S, Burrows R, J Biol Chem 265:14518-25 (1990)).

Suitable linkers may include pH sensitive linkers. For example, certainsuitable linkers may be chosen for their instability in lower pHenvironments to provide for dissociation inside a subcellularcompartment of a target cell. For example, linkers that comprise one ormore trityl groups, derivatized trityl groups, bismaleimideothoxypropane groups, adipic acid dihydrazide groups, and/or acid labiletransferrin groups, may provide for release of components of thecell-targeted molecules of the invention, e.g. a polypeptide component,in environments with specific pH ranges (see e.g. Welhöner H et al., JBiol Chem 266: 4309-14 (1991); Fattom A et al., Infect Immun 60: 584-9(1992)). Certain linkers may be chosen which are cleaved in pH rangescorresponding to physiological pH differences between tissues, such as,e.g., the pH of tumor tissue is lower than in healthy tissues (see e.g.U.S. Pat. No. 5,612,474).

Photocleavable linkers are linkers that are cleaved upon exposure toelectromagnetic radiation of certain wavelength ranges, such as light inthe visible range (see e.g. Goldmacher V et al., Bioconj Chem 3: 104-7(1992)). Photocleavable linkers may be used to release a component of acell-targeted molecule of the invention, e.g. a polypeptide component,upon exposure to light of certain wavelengths. Non-limiting examples ofphotocleavable linkers include a nitrobenzyl group as a photocleavableprotective group for cysteine, nitrobenzyloxycarbonyl chloridecross-linkers, hydroxypropylmethacrylamide copolymer, glycine copolymer,fluorescein copolymer, and methylrhodamine copolymer (Hazum E et al.,Pept Proc Eur Pept Symp, 16th, Brunfeldt K, ed., 105-110 (1981); Senteret al., Photochem Photobiol 42: 231-7 (1985); Yen et al., Makromol Chem190: 69-82 (1989); Goldmacher V et al., Bioconj Chem 3: 104-7 (1992)).Photocleavable linkers may have particular uses in linking components toform cell-targeted molecules of the invention designed for treatingdiseases, disorders, and conditions that can be exposed to light usingfiber optics.

In certain embodiments of the cell-targeted molecules of the presentinvention, a cell-targeting binding region is linked to a CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide usingany number of means known to the skilled worker, including both covalentand noncovalent linkages (see e.g. Chen X et al., Adv Drug Deliv Rev 65:1357-69 (2013); Behrens C, Liu B, MAbs 6: 46-53 (2014).

In certain embodiments of the cell-targeted proteins of the presentinvention, the protein comprises a binding region which is a scFv with alinker connecting a heavy chain variable (V_(H)) domain and a lightchain variable (V_(L)) domain. There are numerous linkers known in theart suitable for this purpose, such as, e.g., the 15-residue (Gly4Ser)₃peptide. Suitable scFv linkers which may be used in forming non-covalentmultivalent structures include GGS, GGGS, GGGGS, GGGGSGGG, GGSGGGG,GSTSGGGSGGGSGGGGSS, and GSTSGSGKPGSSEGSTKG (Plückthun A, Pack P,Immunotechnology 3: 83-105 (1997); Atwell J et al., Protein Eng 12:597-604 (1999); Wu A et al., Protein Eng 14: 1025-33 (2001); Yazaki P etal., J Immunol Methods 253: 195-208 (2001); Carmichael J et al., J MolBiol 326: 341-51 (2003); Amdt M et al., FEBS Lett 578: 257-61 (2004);Bie C et al., World J Hepatol 2: 185-91 (2010)).

Suitable methods for linkage of the components of the cell-targetedmolecules of the present invention may be by any method presently knownin the art for accomplishing such, as long as the attachment does notsubstantially impede the binding capability of the cell-targetingmoiety, the cellular internalization of the CD8+ T-cell hyper-immunizedand/or B-cell/CD4+ T-cell de-immunized polypeptide component, and/orwhen appropriate the desired toxin effector function(s) as measured byan appropriate assay, including assays described herein.

For the purposes of the cell-targeted molecules of the presentinvention, the specific order or orientation is not fixed for thecell-targeting binding region and CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptide region in relation to eachother or the entire cell-targeted molecule (see e.g. FIG. 1). Thecomponents of the polypeptides and cell-targeted molecules of thepresent invention may be arranged in any order provided that the desiredactivities of the cell-targeted moiety and the T-cell hyper-immunizedand/or B-cell/CD4+ T-cell de-immunized effector polypeptide region arenot eliminated. In certain embodiments of the cell-targeted molecules ofthe present invention, the cell-targeting moiety, CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide,and/or endoplasmic reticulum retention/retrieval signal motif may bedirectly linked to each other and/or suitably linked to each other viaone or more intervening polypeptide sequences, such as with one or morelinkers well known in the art and/or described herein.

IV. Examples of Specific Structural Variations of T-Cell EpitopeDelivering, CD8+ T-Cell Hyper-Immunized Polypeptides and Cell-TargetedFusion Proteins Comprising the Same

A T-cell hyper-immunized polypeptide with the capability of delivering aT-cell epitope for MHC class I presentation by a target cell may becreated, in principle, by adding a T-cell epitope to any proteasomedelivering effector polypeptide. A B-cell/CD4+ T-cell de-immunizedsub-variant of the T-cell hyper-immunized polypeptide of the presentinvention may be created by replacing one or more amino acid residues inany B-cell and/or CD4+ T-cell epitope region within a proteasomedelivering effector polypeptide with an overlapping heterologous T-cellepitope. A cell-targeted molecule with the ability to deliver a CD8+T-cell epitope for MHC class I presentation by a target cell may becreated, in principle, by linking any CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptide of the invention to acell-targeting moiety as long as the resulting molecule has a cellularinternalization capability provided by at least the polypeptide of theinvention, the cell-targeting moiety, or the structural combination ofthem together.

A CD8+ T-cell hyper-immunized polypeptide with the capability ofdelivering a CD8+ T-cell epitope for MHC class I presentation by atarget cell may be created by using a toxin-derived, proteasomedelivering effector polypeptide. Similarly, a B-cell/CD4+ T-cellde-immunized, CD8+ T-cell hyper-immunized polypeptide of the presentinvention may be created by replacing one or more amino acid residues inany B-cell epitope region in a toxin-derived, proteasome-deliveringeffector polypeptide with an overlapping heterologous CD8+ T-cellepitope.

Certain T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunizedpolypeptides of the present invention comprise a disruption of at leastone putative B-cell epitope region by the addition of a heterologousT-cell epitope in order to reduce the antigenic and/or immunogenicpotential of the polypeptides after administration to a mammal. Theterms “disrupted” or “disruption” or “disrupting” as used herein withregard to a B-cell epitope region refers to the deletion of at least oneamino acid in a B-cell epitope region, inversion of two or more aminoacids where at least one of the inverted amino acids is in a B-cellepitope region, insertion of at least one amino acid in a B-cell epitoperegion, or mutation of at least one amino acid in a B-cell epitoperegion. A B-cell epitope region disruption by mutation includes aminoacid substitutions with non-standard amino acids and/or non-naturalamino acids. The number of amino acid residues in the region affected bythe disruption is preferably two or more, three or more, four or more,five or more, six or more, seven or more and so forth up to 8, 9, 10,11, 12, or more amino acid residues.

Certain B-cell epitope regions and disruptions are indicated herein byreference to specific amino acid positions of native Shiga toxin ASubunits or a prototypical Diphtheria toxin A Subunit provided in theSequence Listing, noting that naturally occurring toxin A Subunits maycomprise precursor forms containing signal sequences of about 22 aminoacids at their amino-terminals which are removed to produce mature toxinA Subunits and are recognizable to the skilled worker.

Certain T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunizedpolypeptides of the present invention comprise a disruption of at leastone putative CD4+ T-cell epitope region by the addition of aheterologous T-cell epitope in order to reduce the CD4+ T-cell antigenicand/or immunogenic potential of the polypeptides after administration toa mammal. The terms “disrupted” or “disruption” or “disrupting” as usedherein with regard to a CD4+ T-cell epitope region refers to thedeletion of at least one amino acid in a CD4+ T-cell epitope region,inversion of two or more amino acids where at least one of the invertedamino acids is in a CD4+ T-cell epitope, insertion of at least one aminoacid in a CD4+ T-cell epitope region, or mutation of at least one aminoacid in a CD4+ T-cell epitope region. A CD4+ T-cell epitope regiondisruption by mutation includes amino acid substitutions withnon-standard amino acids and/or non-natural amino acids. The number ofamino acid residues in the region affected by the disruption ispreferably two or more, three or more, four or more, five or more, sixor more, seven or more and so forth up to 8, 9, 10, 11, 12, or moreamino acid residues.

Certain CD4+ T-cell epitope regions and disruptions are indicated hereinby reference to specific amino acid positions of native Shiga toxin ASubunits or a prototypical Diphtheria toxin A Subunit provided in theSequence Listing, noting that naturally occurring toxin A Subunits maycomprise precursor forms containing signal sequences of about 22 aminoacids at their amino-terminals which are removed to produce mature toxinA Subunits and are recognizable to the skilled worker.

1. Shiga Toxin Derived, CD8+ T-Cell Epitope Presenting, and B-Cell/CD4+T-Cell De-Immunized Polypeptides

The Shiga toxin family of protein toxins is composed of variousnaturally occurring toxins which are structurally and functionallyrelated, e.g., Shiga toxin, Shiga-like toxin 1, and Shiga-like toxin 2(Johannes L, Romer W, Nat Rev Microbiol 8: 105-16 (2010)). Members ofthe Shiga toxin family share the same overall structure and mechanism ofaction (Engedal, N et al., Microbial Biotech 4: 32-46 (2011)). Forexample, Stx, SLT-1 and SLT-2 display indistinguishable enzymaticactivity in cell free systems (Head S et al., J Biol Chem 266: 3617-21(1991); Tesh V et al., Infect Immun 61: 3392-402 (1993); Brigotti M etal., Toxicon 35:1431-1437 (1997)).

The Shiga toxin family encompasses true Shiga toxin (Stx) isolated fromS. dysenteriae serotype 1, Shiga-like toxin 1 variants (SLT1 or Stx1 orSLT-1 or Slt-I) isolated from serotypes of enterohemorrhagic E. coli,and Shiga-like toxin 2 variants (SLT2 or Stx2 or SLT-2) isolated fromserotypes of enterohemorrhagic E. coli. SLT1 differs by only one residuefrom Stx, and both have been referred to as Verocytotoxins or Verotoxins(VTs) (O'Brien, Curr Top Microbiol Immunol 180: 65-94 (1992)). AlthoughSLT1 and SLT2 variants are only about 53-60% similar to each other atthe amino acid sequence level, they share mechanisms of enzymaticactivity and cytotoxicity common to the members of the Shiga toxinfamily (Johannes, Nat Rev Microbiol 8: 105-16 (2010)). Over 39 differentShiga toxins have been described, such as the defined subtypes Stx1a,Stx1c, Stx1d, and Stx2a-g (Scheutz F et al., J Clin Microbiol 50:2951-63 (2012)). Members of the Shiga toxin family are not naturallyrestricted to any bacterial species because Shiga-toxin-encoding genescan spread among bacterial species via horizontal gene transfer (StrauchE et al., Infect Immun 69: 7588-95 (2001); Zhaxybayeva O, Doolittle W,Curr Biol 21: R242-6 (2011)). As an example of interspecies transfer, aShiga toxin was discovered in a strain of A. haemolyticus isolated froma patient (Grotiuz G et al., J Clin Microbiol 44: 3838-41 (2006)). Oncea Shiga toxin encoding polynucleotide enters a new subspecies orspecies, the Shiga toxin amino acid sequence is presumed to be capableof developing slight sequence variations due to genetic drift and/orselective pressure while still maintaining a mechanism of cytotoxicitycommon to members of the Shiga toxin family (see Scheutz, J ClinMicrobiol 50: 2951-63 (2012)).

For purposes of the present invention, the phrase “Shiga toxin effectorregion” refers to a polypeptide region derived from a Shiga toxin ASubunit of a member of the Shiga toxin family that is capable ofexhibiting at least one Shiga toxin function. Shiga toxin functionsinclude, e.g., cell entry, lipid membrane deformation, directingsubcellular routing, catalytically inactivating ribosomes, effectuatingcytotoxicity, and effectuating cytostatic effects.

For purposes of the present invention, a Shiga toxin effector functionis a biological activity conferred by a polypeptide region derived froma Shiga toxin A Subunit. Non-limiting examples of Shiga toxin effectorfunctions include cellular internalization, subcellular routing,catalytic activity, and cytotoxicity. Non-limiting examples of Shigatoxin catalytic activities include ribosome inactivation, proteinsynthesis inhibition, N-glycosidase activity, polynucleotide:adenosineglycosidase activity, RNAase activity, and DNAase activity. RIPs candepurinate nucleic acids, polynucleosides, polynucleotides, rRNA, ssDNA,dsDNA, mRNA (and polyA), and viral nucleic acids (Barbieri L et al.,Biochem J 286: 1-4 (1992); Barbieri L et al., Nature 372: 624 (1994);Ling J et al., FEBS Lett 345: 143-6 (1994); Barbieri L et al., Biochem J319: 507-13 (1996); Roncuzzi L, Gasperi-Campani A, FEBS Lett 392: 16-20(1996); Stirpe F et al., FEBS Lett 382: 309-12 (1996); Barbieri L etal., Nucleic Acids Res 25: 518-22 (1997); Wang P, Turner N, NucleicAcids Res 27: 1900-5 (1999); Barbieri L et al., Biochim Biophys Acta1480: 258-66 (2000); Barbieri L et al., J Biochem 128: 883-9 (2000);Bagga S et al., J Biol Chem 278: 4813-20 (2003); Picard D et al., J BiolChem 280: 20069-75 (2005)). Some RIPs show antiviral activity andsuperoxide dismutase activity (Erice A et al., Antimicrob AgentsChemother 37: 835-8 (1993); Au T et al., FEBS Lett 471: 169-72 (2000);Parikh B, Turner N, Mini Rev Med Chem 4: 523-43 (2004); Sharma N et al.,Plant Physiol 134: 171-81 (2004)). Shiga toxin catalytic activities havebeen observed both in vitro and in vivo. Assays for Shiga toxin effectoractivity can measure various activities, such as, e.g., proteinsynthesis inhibitory activity, depurination activity, inhibition of cellgrowth, cytotoxicity, supercoiled DNA relaxation activity, and/ornuclease activity.

As used herein, the retention of Shiga toxin effector function refers toa level of Shiga toxin functional activity, as measured by anappropriate quantitative assay with reproducibility comparable to awild-type Shiga toxin effector polypeptide control. For ribosomeinhibition, Shiga toxin effector function is exhibiting an IC₅₀ of10,000 pM or less. For cytotoxicity in a target positive cell killassay, Shiga toxin effector function is exhibiting a CD₅₀ of 1,000 nM orless, depending on the cell type and its expression of the appropriateextracellular target biomolecule.

As used herein, the retention of “significant” Shiga toxin effectorfunction refers to a level of Shiga toxin functional activity, asmeasured by an appropriate quantitative assay with reproducibilitycomparable to a wild-type Shiga toxin effector polypeptide control. Forin vitro ribosome inhibition, significant Shiga toxin effector functionis exhibiting an IC₅₀ of 300 pM or less depending on the source of theribosomes (e.g. bacteria, archaea, or eukaryote (algae, fungi, plants,or animals)). This is significantly greater inhibition as compared tothe approximate IC₅₀ of 100,000 pM for the catalytically inactive SLT-1A1-251 double mutant (Y77S, E167D). For cytotoxicity in a target positivecell kill assay in laboratory cell culture, significant Shiga toxineffector function is exhibiting a CD₅₀ of 100, 50, or 30 nM or less,depending on the cell line and its expression of the appropriateextracellular target biomolecule. This is significantly greatercytotoxicity to the appropriate target cell line as compared to theSLT-1A component alone, without a cell-targeting binding region, whichhas a CD₅₀ of 100-10,000 nM, depending on the cell line.

For some samples, accurate values for either IC₅₀ or CD₅₀ might beunobtainable due to the inability to collect the required data pointsfor an accurate curve fit. Inaccurate IC₅₀ and/or CD₅₀ values should notbe considered when determining significant Shiga toxin effector functionactivity. Data insufficient to accurately fit a curve as described inthe analysis of the data from exemplary Shiga toxin effector functionassays, such as, e.g., assays described in the Examples, should not beconsidered as representative of actual Shiga toxin effector function.For example, theoretically neither an IC₅₀ nor CD₅₀ can be determined ifgreater than 50% ribosome inhibition or cell death, respectively, doesnot occur in a concentration series for a given sample.

The failure to detect activity in Shiga toxin effector function may bedue to improper expression, polypeptide folding, and/or polypeptidestability rather than a lack of cell entry, subcellular routing, and/orenzymatic activity. Assays for Shiga toxin effector functions may notrequire much polypeptide of the invention to measure significant amountsof Shiga toxin effector function activity. To the extent that anunderlying cause of low or no effector function is determinedempirically to relate to protein expression or stability, one of skillin the art may be able to compensate for such factors using proteinchemistry and molecular engineering techniques known in the art, suchthat a Shiga toxin functional effector activity may be restored andmeasured. As examples, improper cell based expression may be compensatedfor by using different expression control sequences; improperpolypeptide folding and/or stability may benefit from stabilizingterminal sequences, or compensatory mutations in non-effector regionswhich stabilize the three dimensional structure of the protein, etc.When new assays for individual Shiga toxin functions become available,CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized Shigatoxin effector polypeptides may be analyzed for any level of those Shigatoxin effector functions, such as a being within 1000-fold or 100-foldor less the activity of a wild-type Shiga toxin effector polypeptide orexhibiting 3-fold to 30-fold or greater activity as compared to afunctional knockout Shiga toxin effector polypeptide.

Sufficient subcellular routing may be merely deduced by observingcytotoxicity in cytotoxicity assays, such as, e.g., cytotoxicity assaysbased on T-cell epitope presentation or based on a toxin effectorfunction involving a cytosolic and/or ER target substrate.

It should be noted that even if the cytotoxicity of a Shiga toxineffector polypeptide is reduced relative to wild-type, in practice,applications using attenuated CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized Shiga toxin effector polypeptides may beequally or more effective than those using wild-type Shiga toxineffector polypeptides because the reduced antigenicity and/orimmunogenicity might offset the reduced cytotoxicity, such as, e.g., byallowing higher dosages, more repeated administrations, or chronicadministration. Wild-type Shiga toxin effector polypeptides are verypotent, being able to kill with only one molecule reaching the cytosolor perhaps 40 molecules being internalized. CD8+ T-cell hyper-immunizedand/or B-cell/CD4+ T-cell de-immunized Shiga toxin effector polypeptideswith even considerably reduced Shiga toxin effector functions, such as,e.g., subcellular routing or cytotoxicity, as compared to wild-typeShiga toxin effector polypeptides may still be potent enough forapplications based on targeted cell killing and/or specific celldetection.

Certain embodiments of the present invention provide polypeptidescomprising a Shiga toxin effector polypeptide comprising an amino acidsequence derived from an A Subunit of a member of the Shiga toxinFamily, the Shiga toxin effector region comprising a disruption of atleast one natively positioned B-cell epitope region provided herein (seee.g. Tables 2, 3, and 4). In certain embodiments, a CD8+ T-cellhyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effectorpolypeptide of the invention may comprise or consist essentially offull-length Shiga toxin A Subunit (e.g. SLT-1A (SEQ ID NO: 1), StxA (SEQID NO:2), or SLT-2A (SEQ ID NO:3)) comprising at least one disruption ofthe amino acid sequence selected from the group of natively positionedamino acids consisting of: the B-cell epitope regions 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 ofSEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48of SEQ ID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ IDNO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 orSEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ IDNO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3;243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ IDNO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQID NO: 1 or SEQ ID NO:2, and the CD4+ T-cell epitope regions 4-33 of SEQID NO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO: 1 or SEQ ID NO:2, 77-103 ofSEQ ID NO:1 or SEQ ID NO:2, 128-168 of SEQ ID NO:1 or SEQ ID NO:2,160-183 of SEQ ID NO: 1 or SEQ ID NO:2, 236-258 of SEQ ID NO: 1 or SEQID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; or the equivalentposition in a conserved Shiga toxin effector polypeptide and/ornon-native Shiga toxin effector polypeptide sequence.

Certain embodiments of the present invention provide polypeptidescomprising a Shiga toxin effector polypeptide comprising an amino acidsequence derived from an A Subunit of a member of the Shiga toxinFamily, the Shiga toxin effector region comprising a disruption of atleast one natively positioned CD4+ T-cell epitope region provided herein(see e.g. Tables 2, 3, and 4). In certain embodiments, a CD8+ T-cellhyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effectorpolypeptide of the invention may comprise or consist essentially offull-length Shiga toxin A Subunit (e.g. SLT-1A (SEQ ID NO: 1), StxA (SEQID NO:2), or SLT-2A (SEQ ID NO:3)) comprising at least one disruption ofthe amino acid sequence selected from the group of natively positionedamino acids consisting of: 4-33, 34-78, 77-103, 128-168, 160-183,236-258, and 274-293; or the equivalent position in a conserved Shigatoxin effector polypeptide and/or non-native Shiga toxin effectorpolypeptide sequence.

In certain embodiments, a Shiga toxin effector polypeptide of thepresent invention may comprise or consist essentially of a truncatedShiga toxin A Subunit. Truncations of Shiga toxin A Subunits mightresult in the deletion of entire B-cell epitope regions withoutaffecting toxin effector catalytic activity and cytotoxicity. Thesmallest Shiga toxin A Subunit fragment exhibiting significant enzymaticactivity is a polypeptide composed of residues 75-247 of StxA (Al-Jaufy,Infect Immun 62: 956-60 (1994)). Truncating the carboxy-terminus ofSLT-1A, StxA, or SLT-2A to amino acids 1-251 removes two predictedB-cell epitope regions, two predicted CD4 positive (CD4+) T-cellepitopes, and a predicted discontinuous B-cell epitope. Truncating theamino-terminus of SLT-1A, StxA, or SLT-2A to 75-293 removes at leastthree predicted B-cell epitope regions and three predicted CD4+ T-cellepitopes. Truncating both amino- and carboxy-terminals of SLT-1A, StxA,or SLT-2A to 75-251 deletes at least five predicted B-cell epitoperegions, four putative CD4+ T-cell epitopes, and one predicteddiscontinuous B-cell epitope.

In certain embodiments, a Shiga toxin effector polypeptide of thepresent invention may comprise or consist essentially of a full-lengthor truncated Shiga toxin A Subunit with at least one mutation, e.g.deletion, insertion, inversion, or substitution, in a provided B-celland/or CD4+ T-cell epitope region. In certain further embodiments, thepolypeptides comprise a disruption which comprises a deletion of atleast one amino acid within the B-cell and/or CD4+ T-cell epitoperegion. In certain further embodiments, the polypeptides comprise adisruption which comprises an insertion of at least one amino acidwithin the B-cell and/or CD4+ T-cell epitope region. In certain furtherembodiments, the polypeptides comprise a disruption which comprises aninversion of amino acids, wherein at least one inverted amino acid iswithin the B-cell and/or CD4+ T-cell epitope region. In certain furtherembodiments, the polypeptides comprise a disruption which comprises amutation, such as an amino acid substitution to a non-standard aminoacid or an amino acid with a chemically modified side chain. Numerousexamples of amino acid substitutions are provided in the Examples.

In other embodiments, the Shiga toxin effector polypeptides of thepresent invention comprises or consists essentially of a truncated Shigatoxin A Subunit which is shorter than a full-length Shiga toxin ASubunit wherein at least one amino acid is disrupted in a nativelypositioned B-cell and/or CD4+ T-cell epitope region provided in theExamples (see Tables 2, 3, and/or 4).

The CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedShiga toxin effector polypeptides of the invention may be smaller thanthe full length A subunit, such as, e.g., consisting of the polypeptideregion from amino acid position 77 to 239 (SLT-1A (SEQ ID NO: 1) or StxA(SEQ ID NO:2)) or the equivalent in other A Subunits of members of theShiga toxin family (e.g. 77 to 238 of (SEQ ID NO:3)). For example, incertain embodiments of the present invention, the Shiga toxin effectorpolypeptides derived from SLT-1A may be derived from amino acids 75 to251 of SEQ ID NO:1, 1 to 241 of SEQ ID NO:1, 1 to 251 of SEQ ID NO:1, oramino acids 1 to 261 of SEQ ID NO:1 wherein at least one amino acid isdisrupted in an endogenous B-cell and/or CD4+ T-cell epitope regionprovided in the Examples (Tables 2, 3, and/or 4). Similarly, CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized Shiga toxineffector regions derived from StxA may comprise or consist essentiallyof amino acids 75 to 251 of SEQ ID NO:2, 1 to 241 of SEQ ID NO:2, 1 to251 of SEQ ID NO:2, or amino acids 1 to 261 of SEQ ID NO:2 wherein atleast one amino acid is disrupted in at least one endogenous B-celland/or CD4+ T-cell epitope region provided in the Examples (Tables 2, 3,and/or 4). Additionally, the Shiga toxin effector regions derived fromSLT-2 may comprise or consist essentially of amino acids 75 to 251 ofSEQ ID NO:3, 1 to 241 of SEQ ID NO:3, 1 to 251 of SEQ ID NO:3, or aminoacids 1 to 261 of SEQ ID NO:3 wherein at least one amino acid isdisrupted in at least one B-cell and/or CD4+ T-cell epitope regionprovided in the Examples (Tables 2, 3, and/or 4).

Certain embodiments of the cell-targeted molecules of the presentinvention each comprise a CD8+ T-cell hyper-immunized and/or B-cell/CD4+T-cell de-immunized Shiga toxin effector polypeptide which retains aShiga toxin effector function but which may be engineered from acytotoxic parental molecule to a polypeptide with diminished orabolished cytotoxicity for non-cytotoxic functions, e.g., effectuatingcytostasis, delivery of exogenous materials, and/or detection of celltypes, by mutating one or more key residues for enzymatic activity.

For certain embodiments, the polypeptides of the present inventioncomprise Shiga toxin effector polypeptides. For certain embodiments, thepolypeptides of the present invention comprise or consist essentially ofone of the polypeptides of SEQ ID NOs: 11-43.

For certain embodiments, the cell-targeted molecules of the presentinvention are cytotoxic proteins comprising Shiga toxin effectorpolypeptides. For certain embodiments, the cell-targeted molecules ofthe present invention comprise or consist essentially of one of thepolypeptides of SEQ ID NOs: 49-54.

2. Diphtheria Toxin Derived, CD8+ T-Cell Hyper-Immunized and/orB-Cell/CD4+ T-Cell De-Immunized Polypeptides

For purposes of the present invention, the phrase “diphtheria toxineffector region” refers to a polypeptide region derived from adiphtheria toxin of a member of the Diphtheria toxin family that iscapable of exhibiting at least one diphtheria toxin function. Diphtheriatoxin functions include, e.g., cell entry, endosome escape, directingsubcellular routing, catalytically inactivating ribosomes, effectuatingcytotoxicity, and effectuating cytostatic effects.

For purposes of the present invention, a diphtheria toxin effectorfunction is a biological activity conferred by a polypeptide regionderived from a diphtheria toxin. Non-limiting examples of diphtheriatoxin effector functions include cellular internalization, subcellularrouting, catalytic activity, and cytotoxicity. Non-limiting examples ofdiphtheria toxin catalytic activities include ribosome inactivation,protein synthesis inhibition, and ADP-ribosylation. Diphtheria toxincatalytic activities have been observed both in vitro and in vivo.Assays for diphtheria toxin effector activity can measure variousactivities, such as, e.g., protein synthesis inhibitory activity,ADP-ribosylation, inhibition of cell growth, and/or cytotoxicity.Sufficient subcellular routing may be merely deduced by observingcytotoxicity in cytotoxicity assays, such as, e.g., cytotoxicity assaysbased on T-cell epitope presentation or based on a toxin effectorfunction involving a cytosolic and/or ER target substrate.

It should be noted that even if a toxin effector activity of adiphtheria toxin effector polypeptide is reduced relative to wild-type,in practice, applications using attenuated CD8+ T-cell hyper-immunizedand/or B-cell/CD4+ T-cell de-immunized diphtheria toxin effectorpolypeptides may be equally or more effective than those usingdiphtheria toxin effector polypeptides with wild-type levels of activitybecause the reduced antigenicity and/or immunogenicity might offset thereduced cytotoxicity, such as, e.g., by allowing higher dosages, morerepeated administrations, or chronic administration. Diphtheria toxineffector polypeptides exhibiting only the effector activity ofsubcellular routing are appropriate for use in applications based ontargeted cell CD8+ T-cell epitope delivery.

Certain embodiments of the present invention provide polypeptidescomprising a diphtheria toxin effector polypeptide comprising an aminoacid sequence derived from an A Subunit of a member of the Diphtheriatoxin Family, the diphtheria toxin effector region comprising adisruption of at least one natively positioned B-cell and/or CD4+ T-cellepitope region provided herein (see e.g. Table 5). In certainembodiments, a CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cellde-immunized diphtheria toxin effector polypeptide of the invention maycomprise or consist essentially of the polypeptide of amino acids 2-389of SEQ ID NO:45 comprising at least one disruption of the amino acidsequence selected from the group of natively positioned amino acidsconsisting of: 3-10 of SEQ ID NO:44, 15-31 of SEQ ID NO:44, 32-54 of SEQID NO:44; 33-43 of SEQ ID NO:44, 71-77 of SEQ ID NO:44, 93-113 of SEQ IDNO:44, 125-131 of SEQ ID NO:44, 138-146 of SEQ ID NO:44, 141-167 of SEQID NO:44, 165-175 of SEQ ID NO:44, 182-201 of SEQ ID NO:45, 185-191 ofSEQ ID NO:44, and 225-238 of SEQ ID NO:45; or the equivalent position ina conserved diphtheria toxin effector polypeptide and/or non-nativediphtheria toxin effector polypeptide sequence.

Optionally, the diphtheria toxin effector polypeptide of the inventionmay comprise one or more mutations (e.g. substitutions, deletions,insertions or inversions) as compared to wild-type as long as at leastone amino acid is disrupted in at least one natively positioned B-celland/or CD4+ T-cell epitope region provided in the Examples (see Table5). In certain embodiments of the invention, the CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized diphtheria toxineffector polypeptides have sufficient sequence identity to a naturallyoccurring diphtheria toxin A Subunit to retain cytotoxicity after entryinto a cell, either by well-known methods of host cell transformation,transfection, infection or induction, or by internalization mediated bya cell-targeting binding region linked with the diphtheria toxineffector polypeptide.

The most critical residues for enzymatic activity and/or cytotoxicity inthe diphtheria toxin A Subunits have been mapped to the followingresidue-positions: histidine-21, tyrosine-27, glycine-52, tryptophan-50,tyrosine-54, tyrosine-65, glutamate-148, and tryptophan-153 (Tweten R etal., J Biol Chem 260: 10392-4 (1985); Wilson B et al., J Biol Chem 269:23296-301 (1994); Bell C, Eisenberg D, Biochemistry 36: 481-8 (1997);Cummings M et al., Proteins 31: 282-98 (1998); Keyvani K et al., LifeSci 64: 1719-24 (1999); Dolan K et al., Biochemistry 39: 8266-75 (2000);Zdanovskaia M et al., Res Micrbiol 151: 557-62 (2000); Kahn K, Bruice T,J Am Chem Soc 123: 11960-9 (2001); Malito E et al., Proc Natl Acad SciUSA 109: 5229-34 (2012)). The capacity of a cytotoxic, cell-targetedmolecule of the invention to cause cell death, e.g. its cytotoxicity,may be measured using any one or more of a number of assays well knownin the art.

Among certain embodiments of the present invention, the polypeptidescomprise the CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cellde-immunized diphteria toxin effector comprising or consistingessentially of amino acids 2 or amino acids 2-389 of SEQ ID NO:45wherein at least one amino acid is disrupted in the natively positionedB-cell epitope and/or CD4+ T-cell epitope regions provided in theExamples (Table 5).

For certain embodiments, the polypeptides of the present inventioncomprise diphtheria toxin effector polypeptides. For certainembodiments, the polypeptides of the present invention comprise orconsist essentially of one of the polypeptides of SEQ ID NOs: 46-48.

For certain embodiments, the cell-targeted molecules of the presentinvention are cytotoxic proteins comprising diphtheria toxin effectorpolypeptides. For certain embodiments, the cell-targeted molecules ofthe present invention comprise or consist essentially of one of thepolypeptides of SEQ ID NOs: 55-60.

For certain embodiments, the polypeptide of the present inventioncomprises or consists essentially of any one of the polypeptides of SEQID NOs: 11-43 or 46-48.

Cell-targeted molecules of the present invention each comprise at leastone T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptide linked to a cell-targeting moiety which can bindspecifically to at least one extracellular target biomolecule inphysical association with a cell, such as a target biomolecule expressedon the surface of a cell. This general structure is modular in that anynumber of diverse cell-targeting moieties may be linked to the CD8+T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides of the present invention.

It is within the scope of the invention to use fragments, variants,and/or derivatives of the polypeptides and cell-targeted molecules ofthe present invention which contain a functional binding site to anyextracellular part of a target biomolecule, and even more preferablycapable of binding a target biomolecule with high affinity (e.g. asshown by K_(D)). Any cell-targeting moiety which binds an extracellularpart of a target biomolecule with a dissociation constant (K_(D)) of10⁻⁵ to 10⁻¹² moles/liter, preferably less than 200 nM, may besubstituted for use in making cell-targeted molecules of the inventionand methods of the invention.

VI. Variations in the Polypeptide Sequence of the T-Cell Hyper-Immunizedand/or B-Cell/CD4+ T-Cell De-Immunized Polypeptides of the Invention andCell-Targeted Molecules Comprising the Same

The skilled worker will recognize that variations may be made to T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides andcell-targeted molecules of the present invention, and polynucleotidesencoding any of the former, without diminishing their biologicalactivities, e.g., by maintaining the overall structure and function ofthe toxin effector region in conjunction with one or more epitopedisruptions which reduce antigenic and/or immunogenic potential. Forexample, some modifications may facilitate expression, purification,and/or pharmacokinetic properties, and/or immunogenicity. Suchmodifications are well known to the skilled worker and include, forexample, a methionine added at the amino terminus to provide aninitiation site, additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons, andbiochemical affinity tags fused to either terminus to provide forconvenient detection and/or purification.

Also contemplated herein is the inclusion of additional amino acidresidues at the amino and/or carboxy termini, such as sequences forepitope tags or other moieties. The additional amino acid residues maybe used for various purposes including, e.g., facilitating cloning,facilitating expression, post-translational modification, facilitatingsynthesis, purification, facilitating detection, and administration.Non-limiting examples of epitope tags and moieties are chitin bindingprotein domains, enteropeptidase cleavage sites, Factor Xa cleavagesites, FIAsH tags, FLAG tags, green fluorescent proteins (GFP),glutathione-S-transferase moieties, HA tags, maltose binding proteindomains, myc tags, polyhistidine tags, ReAsH tags, strep-tags, strep-tagII, TEV protease sites, thioredoxin domains, thrombin cleavage site, andV5 epitope tags.

In certain of the above embodiments, the polypeptide sequence of theCD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides and/or cell-targeted proteins of the invention are variedby one or more conservative amino acid substitutions introduced into thepolypeptide region(s) as long as at least one amino acid is disrupted inat least one natively positioned B-cell epitope region provided herein.As used herein, the term “conservative substitution” denotes that one ormore amino acids are replaced by another, biologically similar aminoacid residue. Examples include substitution of amino acid residues withsimilar characteristics, e.g. small amino acids, acidic amino acids,polar amino acids, basic amino acids, hydrophobic amino acids andaromatic amino acids (see, for example, Table C, infra). An example of aconservative substitution with a residue normally not found inendogenous, mammalian peptides and proteins is the conservativesubstitution of an arginine or lysine residue with, for example,ornithine, canavanine, aminoethylcysteine, or another basic amino acid.For further information concerning phenotypically silent substitutionsin peptides and proteins see, e.g., Bowie J et al., Science 247: 1306-10(1990).

TABLE C Examples of Conservative Amino Acid Substitutions I II III IV VVI VII VIII IX X XI XII XIII XIV A D H C F N A C F A C A A D G E K I W QG M H C D C C E P Q R L Y S I P W F E D D G S N M T L Y G H G E K T V VH K N G P I N P H Q L Q S K R M R T N S R S V Q T T T R V S W P Y T

In the conservative substitution scheme in Table C, exemplaryconservative substitutions of amino acids are grouped by physicochemicalproperties—I: neutral, hydrophilic; II: acids and amides; III: basic;IV: hydrophobic; V: aromatic, bulky amino acids, VI hydrophilicuncharged, VII aliphatic uncharged, VIII non-polar uncharged, IXcycloalkenyl-associated, X hydrophobic, XI polar, XII small, XIIIturn-permitting, and XIV flexible. For example, conservative amino acidsubstitutions include the following: 1) S may be substituted for C; 2) Mor L may be substituted for F; 3) Y may be substituted for M; 4) Q or Emay be substituted for K; 5) N or Q may be substituted for H; and 6) Hmay be substituted for N.

Additional conservative amino acid substitutions include thefollowing: 1) S may be substituted for C; 2) M or L may be substitutedfor F; 3) Y may be substituted for M; 4) Q or E may be substituted forK; 5) N or Q may be substituted for H; and 6) H may be substituted forN.

In certain embodiments, the CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptides and/or cell-targetedmolecules (e.g. cell-targeted proteins) of the present invention maycomprise functional fragments or variants of a polypeptide region of theinvention that have, at most, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid substitutions.

In certain embodiments, the CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptides and/or cell-targetedmolecules of the present invention may comprise functional fragments orvariants of a polypeptide region of the invention that have, at most,20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutionscompared to a polypeptide sequence recited herein, as long as it retainsa disruption of at least one amino acid in a natively positioned B-celland/or CD4+ T-cell epitope region provided in the Examples (Tables 2, 3,4, and/or 5) and as long as the polypeptides or proteins retain a T-cellepitope delivery functionality alone and/or as a component of atherapeutic and/or diagnostic composition. Variants of the CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized Shiga toxineffector polypeptides and/or cell-targeted proteins of the invention arewithin the scope of the invention as a result of changing a polypeptideof the cell-targeted protein of the invention by altering one or moreamino acids or deleting or inserting one or more amino acids, such aswithin the binding region or the CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptide region, in order to achievedesired properties, such as changed cytotoxicity, changed cytostaticeffects, changed immunogenicity, and/or changed serum half-life. AB-cell epitope de-immunized and CD8+ T-cell hyper-immunized polypeptideand/or a cell-targeted protein of the invention may further be with orwithout a signal sequence.

Accordingly, in certain embodiments, the Shiga toxin effector ordiphtheria toxin effector polypeptides of the present invention compriseor consists essentially of amino acid sequences having at least 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99.7%overall sequence identity to a naturally occurring toxin, such as, e.g.,Shiga toxin A Subunit, such as SLT-1A (SEQ ID NO: 1), StxA (SEQ IDNO:2), and/or SLT-2A (SEQ ID NO:3), or a diphtheria toxin catalyticdomain (SEQ ID NO: 44), in certain embodiments, the de-immunized Shigatoxin effector or diphtheria toxin effector polypeptides of the presentinvention comprise or consists essentially of amino acid sequenceshaving at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 99.5% or 99.7% overall sequence identity to a naturally occurringtoxin wherein at least one amino acid is disrupted in at least onenatively positioned B-cell and/or CD4+ T-cell epitope region provided inthe Examples (Tables 2, 3, 4, and/or 5).

In certain embodiments of the cell-targeted molecules of the presentinvention, one or more amino acid residues may be mutated, inserted, ordeleted in order to increase the enzymatic activity of the CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized toxin effectorpolypeptide region. For example, mutating residue-position alanine-231in Stx1A to glutamate increased its enzymatic activity in vitro (SuhanM, Hovde C, Infect Immun 66: 5252-9 (1998)).

In certain embodiments of the cell-targeted molecules of the presentinvention, one or more amino acid residues may be mutated or deleted inorder to reduce or eliminate catalytic and/or cytotoxic activity of theCD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunized toxineffector polypeptide region. For example, the catalytic and/or cytotoxicactivity of the A Subunits of members of the Shiga toxin family orDiphtheria toxin family may be diminished or eliminated by mutation ortruncation.

In certain embodiments of the present invention, the ribotoxin effectorregion has been altered such that it no longer supports catalyticinactivation of a ribosome in vitro. However, other means of modifying aribotoxic effector region to reduce or eliminate ribotoxicity are alsoenvisioned within the scope of the present invention. For example,certain mutations can render a ribotoxic effector region unable to bindits ribosome substrate despite maintaining catalytic ability observableby an in vitro assay whereas other mutations can render a ribotoxicregion unable to target a specific ribonucleic acid sequence within inthe ribosome despite maintaining catalytic ability towards naked nucleicacids in vitro (see e.g. Alford S et al., BMC Biochem 10: 9 (2009);Alvarez-García E et al., Biochim Biophys Act 1814: 1377-82 (2011); WongY et al., PLoS One 7: e49608 (2012)).

In DT, there are several amino acid residues known to be important forcatalytic activity, such as, e.g., histidine-21, tyrosine-27,glycine-52, tryptophan-50, tyrosine-54, tyrosine-65, glutamate-148, andtryptophan-153 (Tweten R et al., J Biol Chem 260: 10392-4 (1985); WilsonB et al., J Biol Chem 269: 23296-301 (1994); Bell C, Eisenberg D,Biochemistry 36: 481-8 (1997); Cummings M et al., Proteins 31: 282-98(1998); Keyvani K et al., Life Sci 64: 1719-24 (1999); Dolan K et al.,Biochemistry 39: 8266-75 (2000); Zdanovskaia M et al., Res Micrbiol 151:557-62 (2000); Kahn K, Bruice T, J Am Chem Soc 123: 11960-9 (2001);Malito E et al., Proc Natl Acad Sci USA 109: 5229-34 (2012)).Glutamate-581 in cholix toxin is conserved with glutamate-148 in DT(Jorgensen R et al., EMBO Rep 9: 802-9 (2008)), and thus, mutations ofglutamate-581 in cholix toxin are predicted to reduce the enzymaticactivity of cholix toxin.

In PE, there are several amino acid residues known to be important forcatalytic activity, such as, e.g., tryptophan-417, histidine-426,histidine-440, glycine-441, arginine-485, tryptophan-458,tryptophan-466, tyrosine-470, tyrosine-481, glutamate-546, arginine-551,glutamate-553, and tryptophan-558 (Douglas C, Collier R, J Bacteriol169: 4967-71 (1987); Wilson B, Colliver R, Curr Top Microbiol Immunol175: 27-41 (1992)); Beattie B et al., Biochemistry 35: 15134-42 (1996);Roberts T, Merrill A, Biochem J 367: 601-8 (2002); Yates S et al.,Biochem J 385: 667-75 (2005); Jorgensen R et al., EMBO Rep 9: 802-9(2008)). Glutamate-574 and glutamate-581 in cholix toxin is conservedwith glutamate-546 and glutamate-553 in PE respectively (Jorgensen R etal., EMBO Rep 9: 802-9 (2008)), and thus, mutations of glutamate-574and/or glutamate-581 in cholix toxin are predicted to reduce theenzymatic activity of cholix toxin.

Because the catalytic domains of cholix toxin, DT, PE, and other relatedenzymes are superimposable (Jorgensen R, et al., J Biol Chem 283:10671-8 (2008)), amino acid residues required for catalytic activity maybe predicted in related polypeptide sequences by sequence alignmentmethods known to the skilled worker.

Several members of the RIP family have been well studied with regard tocatalytic residues. For example, most RIP family members share five keyamino acid residues for catalysis, such as e.g., two tyrosines near theamino terminus of the catalytic domain, a glutamate and arginine nearthe center of the catalytic domain, and a tryptophan near the carboxyterminus of the catalytic domain (Lebeda F, Olson M, Int J Biol Macromol24: 19-26 (1999); Mlsna D et al., Protein Sci 2: 429-35 (1993); deVirgilio M et al., Toxins 2: 2699-737 (2011); Walsh M, Virulence 4:774-84 (2013))). Because the catalytic domains of members of the RIPfamily are superimposable, amino acid residues required for catalyticactivity may be predicted in unstudied and/or new members of the RIPfamily by sequence alignment methods known to the skilled worker (seee.g. Husain J et al., FEBS Lett 342: 154-8 (1994); Ren J et al.,Structure 2: 7-16 (1994); Lebeda F, Olson M, Int J Biol Macromol 24:19-26 (1999); Ma Q et al., Acta Crystallogr D Biol Crystallogr 56: 185-6(2000); Savino C et al., FEBS Lett 470: 239-43 (2000); Robertus J,Monzingo A, Mini Rev Med Chem 4: 477-86 (2004); Mishra V et al., J BiolChem 280: 20712-21 (2005); Zhou C et al., Bioinformatics 21: 3089-96(2005); Lubelli C et al., Anal Biochem 355: 102-9 (2006); Touloupakis Eet al., FEBS J 273: 2684-92 (2006); Hou X et al., BMC Struct Biol 7: 29(2007); Meyer A et al., Biochem Biophys Res Commun 364: 195-200 (2007);Ruggiero A et al., Protein Pept Lett 14: 97-100 (2007); Wang T et al.,Amino Acids 34: 239-43 (2008)).

In the A Subunit of abrin, there are several amino acid residuesimportant for catalytic activity, such as, e.g., tyrosine-74,tyrosine-113, glutamate-164, arginine-167, and tryptophan-198 (Hung C etal., Eur J Biochem 219: 83-7 (1994); Chen J et al., Protein Eng 10:827-33 (1997); Xie L et al., Eur J Biochem 268: 5723-33 (2001)).

In charybdin, there are several amino acid residues important forcatalytic activity, such as, e.g., valine-79, tyrosine-117,glutamate-167, and arginine-170 (Touloupakis E et al., FEBS J 273:2684-92 (2006)).

In the A Subunit of cinnamon, there are several amino acid residuesimportant for catalytic activity, such as, e.g., tyrosine-75,tyrosine-115, glutamate-167, arginine-170, and tryptophan-201 (Hung C etal., Eur J Biochem 219: 83-7 (1994); Chen J et al., Protein Eng 10:827-33 (1997)).

In luffaculin, there are several amino acid residues important forcatalytic activity, such as, e.g., tyrosine-70, glutamate-85,tyrosine-110, glutamate-159, and arginine-162 (Hou X et al., BMC StructBiol 7: 29 (2007)).

In luffins, there are several amino acid residues important forcatalytic activity, such as, e.g., tyrosine-71, glutamate-86,tyrosine-111, glutamate-160, and arginine-163 (Ma Q et al., ActaCrystallogr D Biol Crystallogr 56: 185-6 (2000))

In maize RIPs, there are several amino acid residues important forcatalytic activity, such as, e.g., tyrosine-79, tyrosine-115,glutamate-167, arginine-170, and tryptophan-201 (Robertus J, Monzingo A,Mini Rev Med Chem 4: 477-86 (2004); Yang Y et al., J Mol Biol 395:897-907 (2009)).

In the PD-Ls, there are several amino acid residues important forcatalytic activity, such as, e.g., tyrosine-72, tyrosine-122,glutamate-175, arginine-178, and tryptophan-207 in PDL-1 (Ruggiero A etal., Biopolymers 91: 1135-42 (2009)).

In the A Subunit of the mistletoe RIP, there are several amino acidresidues important for catalytic activity, such as, e.g., tyrosine-66,phenylalanine-75, tyrosine-110, glutamate-159, arginine-162,glutamate-166, arginine-169, and tryptophan-193 (Langer M et al.,Biochem Biophys Res Commun 264: 944-8 (1999); Mishra V et al., ActCrystallogr D Biol Crystallogr 60: 2295-2304 (2004); Mishra V et al., JBiol Chem 280: 20712-21 (2005); Wacker R et al., J Pept Sci 11: 289-302(2005)).

In pokeweed antiviral protein (PAP), there are several amino acidresidues important for catalytic activity, such as, e.g., lysine-48,tyrosine-49, arginine-67, arginine-68, asparagine-69, asparagine-70,tyrosine-72, phenylalanine-90, asparagine-91, aspartate-92,arginine-122, tyrosine-123, glutamate-176, arginine-179, tryptophan-208,and lysine-210 (Rajamohan F et al., J Biol Chem 275: 3382-90 (2000);Rajamohan F et al., Biochemistry 40: 9104-14 (2001)).

In the A chain of ricin, there are several amino acid residues known tobe important for catalytic activity, such as, e.g., arginine-48,tyrosine-80, asparagine-122, tyrosine-123, glutamate-177, arginine-180,serine-203, asparagine-209, tryptophan-211, glycine-212, arginine-213,serine-215, and isoleucine-252 (Frankel A et al., Mol Cell Biol 9:415-20 (1989); Schlossman D et al., Mol Cell Biol 9: 5012-21 (1989);Gould J et al., Mol Gen Genet 230: 91-90 (1991); Ready M et al.,Proteins 10: 270-8 (1991); Rutenber E et al., Proteins 10: 240-50(1991); Monzingo A, Robertus, J, J Mol Biol 227: 1136-45 (1992); Day Pet al., Biochemistry 35: 11098-103 (1996); Marsden C et al., Eur JBiochem 27: 153-62 (2004); Pang Y et al., PLoS One 6: e17883 (2011)). Inricin, there are several amino acid residues which when deleted areknown to impair the catalytic activity of ricin such as, e.g., N24, F25,A28, V29, Y81, V82, V83, G84, E146, E147, A148, I149, S168, F169, I170,I171, C172, I173, Q174, M175, I176, S177, E178, A179, A180, R181, F182,Q183, Y184, D202, P203, I206, T207, N210, S211, W212, and G213(Munishkin A, Wool I, J Biol Chem 270: 30581-7 (1995); Berrondo M, GrayJ, Proteins 79: 2844-60 (2011)).

In saporins, there are several amino acid residues known to be importantfor catalytic activity, such as, e.g., tyrosine-16, tyrosine-72,tyrosine-120, glutamate-176, arginine-179, and tryptophan-208 (Bagga Set al., J Biol Chem 278: 4813-20 (2003); Zarovni N et al., Canc GeneTher 14: 165-73 (2007); Lombardi A et al., FASEB J 24: 253-65 (2010)).In addition, a signal peptide may be included to reduce catalyticactivity (Marshall R et al., Plant J 65: 218-29 (2011)).

In trichosanthins, there are several amino acid residues known to beimportant for catalytic activity, such as, e.g., tyrosine-70,tyrosine-111, glutamate-160, arginine-163, lysine-173, arginine-174,lysine-177, and tryptophan-192 (Wong et al., Eur J Biochem 221: 787-91(1994); Li et al., Protein Eng 12: 999-1004 (1999); Yan et al., Toxicon37: 961-72 (1999); Ding et al., Protein Eng 16: 351-6 (2003); Guo Q etal., Protein Eng 16: 391-6 (2003); Chan D et al., Nucleic Acid Res 35:1660-72 (2007)).

Fungal ribotoxins enzymatically target the same universally conservedSRL ribosomal structure as members of the RIP family and most fungalribotoxins share an RNase Ti type catalytic domain sequence andsecondary structure (Lacadena J et al., FEMS Microbiol Rev 31: 212-37(2007)). Most fungal ribotoxins and related enzymes share three highlyconserved amino acid residues for catalysis, two histidine residues anda glutamate residue (e.g. histidine-40, glutamate-58, and histidine-92in RNase Ti). A DSKKP motif is often present in fungal ribotoxins tospecifically bind the SRL (Kao R, Davies J, J Biol Chem 274: 12576-82(1999)). Because fungal ribotoxin catalytic domains are superimposable,amino acid residues required for catalytic activity may be predicted inunstudied and/or new fungal ribotoxins using one or more sequencealignment methods known to the skilled worker.

For Aspfl, an internal deletion of 16 amino acid residues (positions7-22) severely impaired its ribonucleolytic activity and cytotoxicity(Garciá-Ortega L et al., FEBS J 272: 2536-44 (2005)).

In mitogillin, there are several amino acid residues known to beimportant for catalytic activity, such as, e.g., asparagine-7,histidine-49, glutamate-95, lysine-111, arginine-120, and histidine-136(Kao R et al., Mol Microbiol 29: 1019-27 (1998); Kao R, Davies J, FEBSLett 466: 87-90 (2000)).

In restrictocin, there are several amino acid residues known to beimportant for catalytic activity, such as, e.g., tyrosine-47,histidine-49, glutamate-95, lysine-110, lysine-111, lysine-113,arginine-120, and histidine-136 (Nayak S, Batra J, Biochemistry 36:13693-9 (1997); Nayak S et al., Biochemistry 40: 9115-24 (2001);Plantinga M et al., Biochemistry 50: 3004-13 (2011)).

In α-sarcin, there are several amino acid residues known to be importantfor catalytic activity, such as, e.g., tryptophan-48, histidine-49,histidine-50, tryptophan-51, asparagine-54, isoleucine-69, glutamate-95,glutamate-96, lysine-11, lysine-112, lysine-114, arginine-121,histidine-136, histidine-137, lysine-145 (Lacadena J et al., Biochem J309: 581-6 (1995); Lacadena J et al., Proteins 37: 474-84 (1999);Martínez-Ruiz A et al., Toxicon 37: 1549-63 (1999); de Antonio C et al.,Proteins 41: 350-61 (2000); Masip M et al., Eur J Biochem 268: 6190-6(2001)).

The cytotoxicity of the A Subunits of members of the Shiga toxin familymay be altered, reduced, or eliminated by mutation or truncation. Thepositions labeled tyrosine-77, glutamate-167, arginine-170,tyrosine-114, and tryptophan-203 have been shown to be important for thecatalytic activity of Stx, Stx1, and Stx2 (Hovde C et al., Proc NatlAcad Sci USA 85: 2568-72 (1988); Deresiewicz R et al., Biochemistry 31:3272-80 (1992); Deresiewicz R et al., Mol Gen Genet 241: 467-73 (1993);Ohmura M et al., Microb Pathog 15: 169-76 (1993); Cao C et al.,Microbiol Immunol 38: 441-7 (1994); Suhan M, Hovde C, Infect Immun 66:5252-9 (1998)). Mutating both glutamate-167 and arginine-170 eliminatedthe enzymatic activity of Slt-I A1 in a cell-free ribosome inactivationassay (LaPointe, J Biol Chem 280: 23310-18 (2005)). In another approachusing de novo expression of Slt-I A1 in the endoplasmic reticulum,mutating both glutamate-167 and arginine-170 eliminated Slt-I A1fragment cytotoxicity at that expression level (LaPointe, J Biol Chem280: 23310-18 (2005)). A truncation analysis demonstrated that afragment of StxA from residues 75 to 268 still retains significantenzymatic activity in vitro (Haddad, J Bacteriol 175: 4970-8 (1993)). Atruncated fragment of Slt-I A1 containing residues 1-239 displayedsignificant enzymatic activity in vitro and cytotoxicity by de novoexpression in the cytosol (LaPointe, J Biol Chem 280: 23310-18 (2005)).Expression of a Slt-I A1 fragment truncated to residues 1-239 in theendoplasmic reticulum was not cytotoxic because it could notretrotranslocate to the cytosol (LaPointe, J Biol Chem 280: 23310-18(2005)).

The most critical residues for enzymatic activity and/or cytotoxicity inthe Shiga toxin A Subunits were mapped to the followingresidue-positions: aspargine-75, tyrosine-77, tyrosine-114,glutamate-167, arginine-170, arginine-176, and tryptophan-203 amongothers (Di, Toxicon 57: 535-39 (2011)). In particular, a double-mutantconstruct of Stx2A containing glutamate-E167-to-lysine andarginine-176-to-lysine mutations was completely inactivated; whereas,many single mutations in Stx1 and Stx2 showed a 10-fold reduction incytotoxicity. Further, truncation of Stx1A to 1-239 or 1-240 reduced itscytotoxicity, and similarly, truncation of Stx2A to a conservedhydrophobic residue reduced its cytotoxicity. The most critical residuesfor binding eukaryotic ribosomes and/or eukaryotic ribosome inhibitionin the Shiga toxin A Subunit have been mapped to the followingresidue-positions arginine-172, arginine-176, arginine-179,arginine-188, tyrosine-189, valine-191, and leucine-233 among others(McCluskey A et al., PLoS One 7: e31191 (2012).

Shiga-like toxin 1 A Subunit truncations are catalytically active,capable of enzymatically inactivating ribosomes in vitro, and cytotoxicwhen expressed within a cell (LaPointe, J Biol Chem 280: 23310-18(2005)). The smallest Shiga toxin A Subunit fragment exhibiting fullenzymatic activity is a polypeptide composed of residues 1-239 of Slt1A(LaPointe, J Biol Chem 280: 23310-18 (2005)). Although the smallestfragment of the Shiga toxin A Subunit reported to retain substantialcatalytic activity was residues 75-247 of StxA (Al-Jaufy, Infect Immun62: 956-60 (1994)), a StxA truncation expressed de novo within aeukaryotic cell requires only up to residue 240 to reach the cytosol andexert catalytic inactivation of ribosomes (LaPointe, J Biol Chem 280:23310-18 (2005)).

In certain embodiments of the CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized Shiga toxin effector polypeptides and/orcell-targeted molecules of the present invention derived from SLT-1A(SEQ ID NO: 1) or StxA (SEQ ID NO:2), these changes include substitutionof the asparagine at position 75, tyrosine at position 77, tyrosine atposition 114, glutamate at position 167, arginine at position 170,arginine at position 176, and/or substitution of the tryptophan atposition 203. Examples of such substitutions will be known to theskilled worker based on the prior art, such as asparagine at position 75to alanine, tyrosine at position 77 to serine, substitution of thetyrosine at position 114 to serine, substitution of the glutamateposition 167 to glutamate, substitution of the arginine at position 170to alanine, substitution of the arginine at position 176 to lysine,and/or substitution of the tryptophan at position 203 to alanine. Othermutations which either enhance or reduce Shiga toxin enzymatic activityand/or cytotoxicity are within the scope of the invention and may bedetermined using well known techniques and assays disclosed herein.

The CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides and/or cell-targeted molecules of the invention mayoptionally be conjugated to one or more additional agents, which mayinclude therapeutic and/or diagnostic agents known in the art, includingsuch agents as described herein.

V. General Functions of the CD8+ T-Cell Hyper-Immunized and/orB-Cell/CD4+ T-Cell De-Immunized Polypeptides of the Present Inventionand Cell-Targeted Molecules Comprising the Same

The present invention describes various CD8+ T-cell hyper-immunizedand/or B-cell/CD4+ T-cell de-immunized polypeptides which may be used ascomponents of various compositions of matter, such as cell-targetedcytotoxic molecules and diagnostic compositions. In particular, CD8+T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides have uses as components of various protein therapeutics,such as, e.g. immunotoxins and ligand-toxin fusions, for the targetedkilling of specific cell types for the treatment of a variety ofdiseases, including cancers, immune disorders, and microbial infections.

Any CD8+ T-cell hyper-immunized, polypeptide of the invention may beengineered into a potentially useful, therapeutic, cell-targetedmolecule with the addition of a cell-targeting moiety which targetscellular internalization to a specific cell-type(s) within a chordate,such as, e.g., an amphibian, bird, fish, mammal, reptile, or shark.Similarly, any B-cell epitope de-immunized polypeptide of the inventionmay be engineered into a potentially useful, therapeutic, cell-targetedmolecule with the addition of a cell-targeting moiety which targetscellular internalization to a specific cell-type(s) within a chordate.The present invention provides various cytotoxic cell-targeted moleculescomprising CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cellde-immunized polypeptides functionally associated with binding regionsto effectuate cell targeting such that the cytotoxic cell-targetedmolecules selectively delivery T-cell epitopes, kill, inhibit the growthof, deliver exogenous material to, and/or detect specific cell types.This system is modular, in that any number of diverse binding regionsmay be used to target to diverse cell types any CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide ofthe invention, including.

The presentation of a T-cell immunogenic epitope peptide by the MHCclass I complex targets the presenting cell for killing by CTL-mediatedcytolysis. By engineering MHC class I peptides into proteasomedelivering effector polypeptide components of target-cell-internalizingtherapeutics, the targeted delivery and presentation ofimmuno-stimulatory antigens may be accomplished by harnessing vertebratetarget cells' endogenous MHC class I pathways. The presentation bytargeted cells of immuno-stimulatory non-self antigens, such as, e.g.,known viral epitope-peptides with high immunogenicity, can signal toother immune cells to destroy the target cells and recruit more immunecells to the target cell site within an organism.

Thus, already cytotoxic molecules, such as e.g. potential therapeuticscomprising cytotoxic toxin effector regions, may be engineered usingmethods of the invention into more cytotoxic molecules and/or to have anadditional cytotoxicity mechanism operating via effector T-cells. Thesemultiple cytotoxic mechanisms may complement each other (such as byproviding both direct target cell killing and indirect (CTL-mediated)cell killing, redundantly backup each other (such as by providing onemechanism of cell killing in the absence of the other), and/or protectagainst the development of therapeutic resistance (by limitingresistance to the less probable situation of the malignant or infectedcell evolving to block two different cell-killing mechanismssimultaneously).

In addition, parental cytotoxic molecules which rely on toxin and/orenzymatic regions for cytotoxicity may be engineered to be cytotoxiconly via T-cell epitope cytosolic delivery and presentation by bothembedding a T-cell epitope and inactivating the enzymatic activity ofthe parental molecule, either with the embedded T-cell epitope orindependently by other means such as mutation or truncation. Thisapproach removes one cytotoxic mechanism while adding another and addsthe capability of immuno-stimulation to the local area. Furthermore,parental cytotoxic molecules which rely on toxin and/or enzymaticregions for cytotoxicity may be engineered to be cytotoxic only viaT-cell epitope cytosolic delivery and presentation by embedding a T-cellepitope in the enzymatic domain of the parental molecule such that theenzymatic activity is reduced or eliminated by the sequence changes thatcreate the heterologous T-cell epitope. This allows for the one-stepmodification of enzymatically-cytotoxic molecules, which have theability to internalize into cells and route to the cytosol, intoenzymatically inactive, cytotoxic molecules which rely on T-cell epitopeproteasome delivery and presentation for cytotoxicity and localimmuno-stimulation.

A. Delivery of T-Cell Epitopes for MHC Class I Presentation on a CellSurface

One function of certain CD8+ T-cell hyper-immunized polypeptides andcell-targeted molecules of the present invention is the delivery of oneor more T-cell epitopes for MHC class I presentation by a cell. Deliveryof exogenous T-cell epitope peptides to the MHC class I system of atarget cell can be used to induce the target cell to present the T-cellepitope peptide in association with MHC class I molecules on the cellsurface, which subsequently leads to the activation of CD8+ effectorT-cells to attack the target cell.

Certain embodiments of the CD8+ T-cell hyper-immunized polypeptides andcell-targeted molecules of the present invention are capable ofdelivering one or more T-cell epitopes to the proteasome of a targetcell. The delivered T-cell epitope are then proteolytic processed andpresented by the MHC class I pathway on the outside surface of thetarget cell.

The applications of these T-cell epitope presenting functions of theCD8+ T-cell hyper-immunized polypeptides and cell-targeted molecules ofthe present invention are vast. Every nucleated cell in a mammalianorganism may be capable of MHC class I pathway presentation ofimmunogenic T-cell epitope peptides on their cell outer surfacescomplexed to MHC class I molecules. In addition, the sensitivity ofT-cell epitope recognition is so exquisite that only a few MHC-I peptidecomplexes are required to be presented—even presentation of a singlecomplex can be sufficient for recognition by an effector T-cell (SykulevY et al., Immunity 4: 565-71 (1996)).

In order for a heterologous T-cell epitope to be presented on a targetcell surface, the polypeptide delivering the heterologous T-cellepitope-peptide must be degraded by a proteasome in the target cell suchthat a peptide fragment comprising the T-cell epitope is created andtransported to the lumen of the ER for loading onto a MHC class Imolecule.

In addition, the CD8+ T-cell hyper-immunized polypeptide must firstreach the interior of a target cell and then come in contact with aproteasome in the target cell. In order to deliver a CD8+ T-cellhyper-immunized polypeptide of the present invention to the interior ofa target cell, cell-targeting molecules of the present invention must becapable of target cell internalization. Once the CD8+ T-cellhyper-immunized polypeptide of the invention is internalized as acomponent of a cell-targeting molecule, the CD8+ T-cell hyper-immunizedpolypeptide will typical reside in an early endosomal compartment, suchas, e.g., endocytotic vesicle. The CD8+ T-cell hyper-immunizedpolypeptide then has to reach a target cell's proteasome with at leastone intact, heterologous T-cell epitope.

These functions can be detected and monitored by a variety of standardmethods known in the art to the skilled worker. For example, the abilityof cell-targeted molecules of the present invention to deliver a T-cellepitope peptide and drive presentation of the epitope peptide by the MHCclass I system of target cells may be investigated using various invitro and in vivo assays, including, e.g., the directdetection/visualization of MHC class I/peptide complexes, measurement ofbinding affinities for the heterologous T-cell epitope peptide to MHCClass I molecules, and/or measurement of functional consequences of MHCclass I-epitope peptide complex presentation on target cells bymonitoring CTL responses.

Certain assays to monitor this function of the polypeptides andmolecules of the present invention involve the direct detection of aspecific MHC Class I/peptide antigen complex in vitro or ex vivo. Commonmethods for direct visualization and quantitation of peptide-MHC class Icomplexes involve various immuno-detection reagents known to the skilledworker. For example, specific monoclonal antibodies can be developed torecognize a particular MHC/Class I/peptide antigen complex (Porgador Aet al, Immunity 6: 715-26 (1997)). Similarly, soluble, multimeric T cellreceptors, such as the TCR-STAR reagents (Altor, Mirmar, Fla., U.S.) canbe used to directly visualize or quantitate specific MHC I/antigencomplexes (Zhu X et al., J Immunol 176: 3223-32 (2006)). These specificmAbs or soluble, multimeric T-cell receptors may be used with variousdetection methods, including, e.g. immunohistochemistry, flow cytometry,and enzyme-linked immuno assay (ELISA).

An alternative method for direct identification and quantification ofMHC I/peptide complexes involves mass spectrometry analyses, such as,e.g., the ProPresent Antigen Presentation Assay (ProImmune, Inc.,Sarasota, Fla., U.S.) in which peptide-MCH class I complexes areextracted from the surfaces of cells, then the peptides are purified andidentified by sequencing mass spectrometry (Falk K et al., Nature 351:290-6 (1991)).

Certain assays to monitor the T-cell epitope delivery and MHC class Ipresentation function of the polypeptides and molecules of the presentinvention involve computational and/or experimental methods to monitorMHC Class I and peptide binding and stability. Several software programsare available for use by the skilled worker for predicting the bindingresponses of epitope peptides to MHC Class I alleles, such as, e.g., TheImmune Epitope Database and Analysis Resource (IEDB) Analysis ResourceMHC-I binding prediction Consensus tool (Kim Y et al., Nucleic Acid Res40: W525-30 (2012). Several experimental assays have been routinelyapplied, such as, e.g. cell surface binding assays and/or surfaceplasmon resonance assays to quantify and/or compare binding kinetics(Miles K et al., Mol Immunol 48: 728-32 (2011)). Additionally, otherMHC-peptide binding assays based on a measure of the ability of apeptide to stabilize the ternary MHC-peptide complex for a given MHCClass I allele, as a comparison to known controls, have been developed(e.g., MHC-peptide binding assay from ProImmmune, Inc.).

Alternatively, measurements of the consequence of MHC Class I/peptideantigen complex presentation on the cell surface can be performed bymonitoring the cytotoxic T cell (CTL) response to the specific complex.These measurements by include direct labeling of the CTLs with MHC ClassI tetramer or pentamer reagents. Tetramers or pentamers bind directly toT cell receptors of a particular specificity, determined by the MajorHistocompatibility Complex (MHC) allele and peptide combination.Additionally, the quantification of released cytokines, such asinterferon gamma or interleukins by ELISA or enzyme-linked immunospot(ELIspot) is commonly assayed to identify specific CTL responses. Thecytotoxic capacity of CTL can be measured using a number of assays,including the classical 51 Chromium (Cr) release assay or alternativenon-radioactive cytotoxicity assays (e.g., CytoTox96® non-radioactivekits and CellTox™ CellTiter-GLO® kits available from Promega Corp.,Madison, Wis., U.S.), Granzyme B ELISpot, Caspase Assays or LAMP-1translocation flow cytometric assays. To specifically monitor thekilling of target cells, Carboxyfluorescein diacetate succinimidyl ester(CFSE) can be used to easily and quickly label a cell population ofinterest for in vitro or in vivo investigation to monitor killing ofepitope specific CSFE labeled target cells (Durward M et al., J Vis Exp45 pii 2250 (2010)).

In vivo responses to MHC Class I presentation can be followed byadministering a MHC Class I/antigen promoting agent (e.g., a peptide,protein or inactivated/attenuated virus vaccine) followed by challengewith an active agent (e.g. a virus) and monitoring responses to thatagent, typically in comparison with unvaccinated controls. Ex vivosamples can be monitored for CTL activity with methods similar to thosedescribed previously (e.g. CTL cytotoxicity assays and quantification ofcytokine release).

HLA-A, HLA-B, and/or HLA-C molecules are isolated from the intoxicatedcells after lysis using immune affinity (e.g., an anti-MHC antibody“pulldown” purification) and the associated peptides (i.e., the peptidespresented by the isolated MHC molecules) are recovered from the purifiedcomplexes. The recovered peptides are analyzed by sequencing massspectrometry. The mass spectrometry data is compared against a proteindatabase library consisting of the sequence of the exogenous (non-self)peptide (T-cell epitope X) and the international protein index forhumans (representing “self” or non-immunogenic peptides). The peptidesare ranked by significance according to a probability database. Alldetected antigenic (non-self) peptide sequences are listed. The data isverified by searching against a scrambled decoy database to reduce falsehits (see e.g. Ma B, Johnson R, Mol Cell Proteomics 11: O111.014902(2012)). The results will demonstrate that peptides from the T-cellepitope X are presented in MHC complexes on the surface of intoxicatedtarget cells.

The set of presented peptide-antigen-MHC complexes can vary betweencells due to the antigen-specific HLA molecules expressed. T-cells canthen recognize specific peptide-antigen-MHC complexes displayed on acell surface using different TCR molecules with differentantigen-specificities.

Because multiple T-cell epitopes may be delivered by a cell-targetedmolecule of the invention, such as, e.g., by embedding two or moredifferent T-cell epitopes in a single proteasome delivering effectorpolypeptide, a single cell-targeted molecule of the invention may beeffective chordates of the same species with different MHC classvariants, such as, e.g., in humans with different HLA alleles. This mayallow for the simultaneously combining different T-cell epitopes withdifferent effectiveness in different sub-populations of subjects basedon MHC complex protein diversity and polymorphisms (see e.g. Yuhki N etal., J Hered 98: 390-9 (2007)). For example, human MHC complex proteins,HLA proteins, vary among humans based on genetic ancestry, e.g. African(sub-Saharan), Amerindian, Caucasiod, Mongoloid, New Guinean andAustralian, or Pacific islander (see e.g. Wang M, Claesson M, MethodsMol Biol 1184: 309-17 (2014)).

The activation of T-cell responses are desired characteristics ofcertain anti-cancer, anti-neoplastic, anti-tumor, and/or anti-microbialbiologic drugs to stimulate the patient's own immune system towardtargeted cells. Activation of a robust and strong T-cell response isalso a desired characteristic of many vaccines (Aly H A, J ImmunolMethods 382: 1-23 (2012)). The presentation of a T-cell epitope by atarget cell within an organism can lead to the activation of robustimmune responses to a target cell and/or its general locale within anorganism. Thus, the targeted delivery of a T-cell epitope forpresentation may be utilized for engineering the activation of T-cellresponses during a therapeutic regime.

B. Cell Kill Via Targeted Cytotoxicity and/or Recruitment of CTLs

Cell-targeted molecules of the present invention comprising CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides ofthe present invention can provide both: 1) cell type specificT-cell-epitope delivery for MHC class I presentation and 2) potentcytotoxicity. In addition, certain embodiments of the cell-targetedmolecules of the present invention also provide de-immunization, whichreduces the likelihood of certain immune responses when administered toa mammal.

In certain embodiments of the cell-targeted molecules of the presentinvention, upon contacting a cell physically coupled with anextracellular target biomolecule of the cell-targeting moiety (e.g. acell-targeted binding region), the cell-targeted molecule of theinvention is capable of causing death of the cell. The mechanism of cellkill may be direct, e.g. via the enzymatic activity of a toxin effectorregion, or indirect via CTL-mediated cytolysis, and may be under variedconditions of target cells, such as an ex vivo manipulated target cell,a target cell cultured in vitro, a target cell within a tissue samplecultured in vitro, or a target cell in vivo.

1. Indirect Cell Kill Via T-Cell Epitope Delivery and MHC Class IPresentation

T-cell epitope delivering, CD8+ T-cell hyper-immunized polypeptides ofthe present invention, with or without B-cell epitope de-immunization,may be used as components of cell-targeted molecules for indirect cellkill. Certain embodiments of the cell-targeted molecules of the presentinvention are cytotoxic because they comprise a CD8+ T-cell epitopepresenting polypeptide of the invention which delivers one or moreT-cell epitopes to the MHC class I presentation pathway of a target cellupon target internalization of the cell-targeted molecule.

In certain embodiments of the cell-targeted molecules of the presentinvention, upon contacting a cell physically coupled with anextracellular target biomolecule of the cell-targeting moiety (e.g. acell-targeted binding region), the cell-targeted molecule of theinvention is capable of indirectly causing the death of the cell, suchas, e.g., via the presentation of one or more T-cell epitopes by thetarget cell and the subsequent recruitment of CTLs.

2. Direct Cell Kill Via Cell-Targeted Toxin Cytotoxicity

T-cell epitope delivering, CD8+ T-cell hyper-immunized, and/orB-cell/CD4+ T-cell de-immunized polypeptides of the present inventionmay be used as components of cell-targeted molecules for direct cellkill.

Because many naturally occurring toxins are adapted to killingeukaryotic cells, cytotoxic proteins designed using toxin-derived,proteasome delivering effector regions, can show potent cell-killactivity. In particular, proteasome delivering effector regions may alsocomprise ribotoxic toxin effector polypeptides. However, other toxineffector regions are contemplated for use in the cell-targeted moleculesof the invention, such as, e.g., polypeptides from toxins which do notcatalytically inactive ribosomes but rather are cytotoxic due to othermechanisms. For example, cholix toxins, heat-labile enterotoxins, andpertussis toxins heterotrimeric G proteins by attacking the Gsalphasubunit.

The A Subunits of many members of the ABx toxin superfamily compriseenzymatic domains capable of killing a eukaryotic cell once in thecell's cytosol. The replacement of a B-cell epitope with a T-cellepitope within multiple ABx toxin-derived, polypeptides comprising toxinenzymatic domains did not significantly alter their enzymatic activity.Thus, the CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cellde-immunized polypeptides of the present invention can potentiallyprovide two mechanisms of cell kill.

Certain embodiments of the cell-targeted molecules of the presentinvention are cytotoxic because they comprise a CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide ofthe invention which comprises an active toxin component.

In certain embodiments of the cell-targeted molecules of the presentinvention, upon contacting a cell physically coupled with anextracellular target biomolecule of the cell-targeting moiety (e.g. acell-targeted binding region), the cell-targeted molecule of theinvention is capable of directly causing the death of the cell, such as,e.g., via the enzymatic activity of a toxin effector region.

C. De-Immunization Improves Applications Involving Administration toMammals

The polypeptides and cell-targeted molecules of the present inventionhave improved usefulness for administration to mammalian species aseither a therapeutic and/or diagnostic agent because of the reducedlikelihood of producing undesired immune responses in mammals whileincreasing the likelihood of producing desirable immune responses inmammals.

Certain CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cellde-immunized toxin-derived polypeptides of the present invention mightdiffer in their antigenicity profiles when administered to variousmammals, but are expected to have reduced B-cell and/or CD4+ T-cellantigenicity and/or immunogenicity. In certain embodiments, the desiredbiological functions of the original toxin polypeptide from which thede-immunized CD8+ T-cell hyper-immunized polypeptide was derived arepreserved in the polypeptides of the invention after the B-cellepitope(s) was disrupted and the CD8+ T-cell epitope was added. Inaddition, B-cell epitopes often coincide or overlap with epitopes ofmature CD4+ T-cells, thus the disruption of a B-cell epitope oftensimultaneously disrupts a CD4+ T-cell epitope.

D. Selective Cytotoxicity Among Cell Types

Certain cell-targeted molecules of the present invention have uses inthe selective killing of specific target cells in the presence ofuntargeted, bystander cells. By targeting the delivery of immunogenicT-cell epitopes to the MHC class I pathway of target cells, thesubsequent presentation of T-cell epitopes and CTL-mediated cytolysis oftarget cells induced by the cell-targeted molecules of the invention canbe restricted to preferentially killing selected cell types in thepresence of untargeted cells. In addition, the killing of target cellsby the potent cytotoxic activity of various toxin effector regions canbe restricted to preferentially killing target cells with thesimultaneous delivery of an immunogenic T-cell epitope and a cytotoxictoxin effector polypeptide.

In certain embodiments, upon administration of the cell-targetedmolecule of the present invention to a mixture of cell types, thecell-targeted molecule is capable of selectively killing those cellswhich are physically coupled with an extracellular target biomoleculecompared to cell types not physically coupled with an extracellulartarget biomolecule. Because many toxins are adapted for killingeukaryotic cells, such as, e.g., members of the ABx and ribotoxinfamilies, cytotoxic proteins designed using toxin effector regions canshow potent cytotoxic activity. By targeting the delivery ofenzymatically active toxin effector regions to specific cell types usinghigh-affinity binding regions, this potent cell kill activity can berestricted to killing only those cell types desired to be targeted bytheir physical association with a target biomolecule of the chosenbinding regions.

In certain embodiments, the cytotoxic, cell-targeted molecule of thepresent invention is capable of selectively or preferentially causingthe death of a specific cell type within a mixture of two or moredifferent cell types. This enables the targeted cytotoxic activity tospecific cell types with a high preferentiality, such as a 3-foldcytotoxic effect, over “bystander” cell types that do not express thetarget biomolecule. Alternatively, the expression of the targetbiomolecule of the binding region may be non-exclusive to one cell typeif the target biomolecule is expressed in low enough amounts and/orphysically coupled in low amounts with cell types that are not to betargeted. This enables the targeted cell-killing of specific cell typeswith a high preferentiality, such as a 3-fold cytotoxic effect, over“bystander” cell types that do not express significant amounts of thetarget biomolecule or are not physically coupled to significant amountsof the target biomolecule.

In certain further embodiments, upon administration of the cytotoxiccell-targeted molecule to two different populations of cell types, thecytotoxic cell-targeted molecule is capable of causing cell death asdefined by the half-maximal cytotoxic concentration (CD₅₀) on apopulation of target cells, whose members express an extracellulartarget biomolecule of the binding region of the cytotoxic protein, at adose at least three-times lower than the CD₅₀ dose of the same cytotoxicprotein to a population of cells whose members do not express anextracellular target biomolecule of the binding region of the cytotoxicprotein.

In certain embodiments, the cytotoxic activity toward populations ofcell types physically coupled with an extracellular target biomoleculeis at least 3-fold higher than the cytotoxic activity toward populationsof cell types not physically coupled with any extracellular targetbiomolecule of the binding region. According to the present invention,selective cytotoxicity may be quantified in terms of the ratio (a/b) of(a) cytotoxicity towards a population of cells of a specific cell typephysically coupled with a target biomolecule of the binding region to(b) cytotoxicity towards a population of cells of a cell type notphysically coupled with a target biomolecule of the binding region. Incertain embodiments, the cytotoxicity ratio is indicative of selectivecytotoxicity which is at least 3-fold, 5-fold, 10-fold, 15-fold,20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold,250-fold, 500-fold, 750-fold, or 1000-fold higher for populations ofcells or cell types physically coupled with a target biomolecule of thebinding region compared to populations of cells or cell types notphysically coupled with a target biomolecule of the binding region.

This preferential cell-killing function allows a targeted cell to bekilled by certain cytotoxic, cell-targeted molecules of the presentinvention under varied conditions and in the presence of non-targetedbystander cells, such as ex vivo manipulated mixtures of cell types, invitro cultured tissues with mixtures of cell types, or in vivo in thepresence of multiple cell types (e.g. in situ or in a native locationwithin a multicellular organism).

E. Delivery of Additional Exogenous Material into the Interior ofTargeted Cells

In addition to cytotoxic and cytostatic applications, cell-targetedmolecules of the present invention optionally may be used forinformation gathering and diagnostic functions. Further, non-toxicvariants of the cytotoxic, cell-targeted molecules of the invention, oroptionally toxic variants, may be used to deliver additional exogenousmaterials to and/or label the interiors of cells physically coupled withan extracellular target biomolecule of the cytotoxic protein. Varioustypes of cells and/or cell populations which express target biomoleculesto at least one cellular surface may be targeted by the cell-targetedmolecules of the invention for receiving exogenous materials. Thefunctional components of the present invention are modular, in thatvarious toxin effector regions and additional exogenous materials may belinked to various binding regions to provide diverse applications, suchas non-invasive in vivo imaging of tumor cells.

Because the cell-targeted molecules of the invention, including nontoxicforms thereof, are capable of entering cells physically coupled with anextracellular target biomolecule recognized by the cell-targetedmolecule's binding region, certain embodiments of the cell-targetedmolecules of the invention may be used to deliver additional exogenousmaterials into the interior of targeted cell types. In one sense, theentire cell-targeted molecule of the invention is an exogenous materialwhich will enter the cell; thus, the “additional” exogenous materialsare heterologous materials linked to but other than the corecell-targeted molecule itself. CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptides which become non-toxicafter T-cell epitope addition may still be useful for deliveringexogenous materials into cells (e.g. T-cell epitope replacementsoverlapping amino acid resides critical for catalytic function of atoxin effector region).

“Additional exogenous material” as used herein refers to one or moremolecules, often not generally present within a native target cell,where the proteins of the present invention can be used to specificallytransport such material to the interior of a cell. Non-limiting examplesof additional exogenous materials are peptides, polypeptides, proteins,polynucleotides, small molecule chemotherapeutic agents, and detectionpromoting agents.

In certain embodiments, the additional exogenous material comprises aprotein or polypeptide comprising an enzyme. In certain otherembodiments, the additional exogenous material is a nucleic acid, suchas, e.g. a ribonucleic acid that functions as a small inhibiting RNA(siRNA) or microRNA (miRNA). In certain embodiments, the additionalexogenous material is an antigen, such as antigens derived frombacterial proteins, viral proteins, proteins mutated in cancer, proteinsaberrantly expressed in cancer, or T-cell complementary determiningregions. For example, exogenous materials include antigens, such asthose characteristic of antigen-presenting cells infected by bacteria,and T-cell complementary determining regions capable of functioning asexogenous antigens. Additional examples of exogenous materials includepolypeptides and proteins larger than an antigenic peptide, such asenzymes. Exogenous materials comprising polypeptides or proteins mayoptionally comprise one or more antigens whether known or unknown to theskilled worker.

F. Information Gathering for Diagnostic Functions

Certain cell-targeted molecules of the present invention have uses inthe in vitro and/or in vivo detection of specific cells, cell types,and/or cell populations. In certain embodiments, the proteins describedherein are used for both diagnosis and treatment, or for diagnosisalone. When the same cytotoxic protein is used for both diagnosis andtreatment, the cytotoxic protein variant which incorporates a detectionpromoting agent for diagnosis may be rendered nontoxic by catalyticinactivation of a toxin effector region via one or more amino acidsubstitutions, including exemplary substitutions described herein.Nontoxic forms of the cytotoxic, cell-targeted molecules of theinvention that are conjugated to detection promoting agents optionallymay be used for diagnostic functions, such as for companion diagnosticsused in conjunction with a therapeutic regimen comprising the same or arelated binding region.

The ability to conjugate detection promoting agents known in the art tovarious cell-targeted molecules of the present invention provides usefulcompositions for the detection of cancer, tumor, immune, and infectedcells. These diagnostic embodiments of the cell-targeted molecules ofthe invention may be used for information gathering via various imagingtechniques and assays known in the art. For example, diagnosticembodiments of the cell-targeted molecules of the invention may be usedfor information gathering via imaging of intracellular organelles (e.g.endocytotic, Golgi, endoplasmic reticulum, and cytosolic compartments)of individual cancer cells, immune cells, or infected cells in a patientor biopsy sample.

Various types of information may be gathered using the diagnosticembodiments of the cell-targeted molecules of the invention whether fordiagnostic uses or other uses. This information may be useful, forexample, in diagnosing neoplastic cell types, determining therapeuticsusceptibilities of a patient's disease, assaying the progression ofanti-neoplastic therapies over time, assaying the progression ofimmunomodulatory therapies over time, assaying the progression ofantimicrobial therapies over time, evaluating the presence of infectedcells in transplantation materials, evaluating the presence of unwantedcell types in transplantation materials, and/or evaluating the presenceof residual tumor cells after surgical excision of a tumor mass.

For example, subpopulations of patients might be ascertained usinginformation gathered using the diagnostic variants of the cell-targetedmolecules of the invention, and then individual patients could becategorized into subpopulations based on their unique characteristic(s)revealed using those diagnostic embodiments. For example, theeffectiveness of specific pharmaceuticals or therapies might be one typeof criterion used to define a patient subpopulation. For example, anontoxic diagnostic variant of a particular cytotoxic, cell-targetedmolecule of the invention may be used to differentiate which patientsare in a class or subpopulation of patients predicted to respondpositively to a cytotoxic variant of the same cell-targeted molecule ofthe invention. Accordingly, associated methods for patientidentification, patient stratification, and diagnosis using CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized cell-targetedmolecules of the present invention, including non-toxic variants ofcytotoxic, cell-targeted molecules of the present invention, areconsidered to be within the scope of the present invention.

VII. Production, Manufacture, and Purification of CD8+ T-CellHyper-Immunized and/or B-Cell/CD4+ T-Cell De-Immunized Polypeptides ofthe Present Invention and the Cell-Targeted Molecules Comprising theSame

The CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides and cell-targeted molecules of the present invention may beproduced using biochemical engineering techniques well known to those ofskill in the art. For example, polypeptides and cell-targeted moleculesof the invention may be manufactured by standard synthetic methods, byuse of recombinant expression systems, or by any other suitable method.Thus, polypeptides and cell-targeted proteins of the present inventionmay be synthesized in a number of ways, including, e.g. methodscomprising: (1) synthesizing a polypeptide or polypeptide component of aprotein using standard solid-phase or liquid-phase methodology, eitherstepwise or by fragment assembly, and isolating and purifying the finalpeptide compound product; (2) expressing a polynucleotide that encodes apolypeptide or polypeptide component of a cell-targeted protein of theinvention in a host cell and recovering the expression product from thehost cell or host cell culture; or (3) cell-free in vitro expression ofa polynucleotide encoding a polypeptide or polypeptide component of acell-targeted protein of the invention, and recovering the expressionproduct; or by any combination of the methods of (1), (2) or (3) toobtain fragments of the peptide component, subsequently joining (e.g.ligating) the fragments to obtain the peptide component, and recoveringthe peptide component.

It may be preferable to synthesize a CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptide or a protein or polypeptidecomponent of a cell-targeted protein of the invention by means ofsolid-phase or liquid-phase peptide synthesis. Polypeptides andcell-targeted molecules of the present invention may suitably bemanufactured by standard synthetic methods. Thus, peptides may besynthesized by, e.g. methods comprising synthesizing the peptide bystandard solid-phase or liquid-phase methodology, either stepwise or byfragment assembly, and isolating and purifying the final peptideproduct. In this context, reference may be made to WO 1998/11125 or,inter alia, Fields G et al., Principles and Practice of Solid-PhasePeptide Synthesis (Synthetic Peptides, Grant G, ed., Oxford UniversityPress, U.K., 2nd ed., 2002) and the synthesis examples therein.

CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides and cytotoxic, cell-targeted proteins of the presentinvention may be prepared (produced and purified) using recombinanttechniques well known in the art. In general, methods for preparingpolypeptides by culturing host cells transformed or transfected with avector comprising the encoding polynucleotide and recovering thepolypeptide from cell culture are described in, e.g. Sambrook J et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, NY, U.S., 1989); Dieffenbach C et al., PCR Primer: A LaboratoryManual (Cold Spring Harbor Laboratory Press, N.Y., U.S., 1995). Anysuitable host cell may be used to produce a polypeptide and/orcell-targeted protein of the invention. Host cells may be cells stablyor transiently transfected, transformed, transduced or infected with oneor more expression vectors which drive expression of a polypeptide ofthe invention. In addition, a CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptides and/or cell-targetedprotein of the invention may be produced by modifying the polynucleotideencoding a polypeptide or cell-targeted protein of the invention thatresult in altering one or more amino acids or deleting or inserting oneor more amino acids in order to achieve desired properties, such aschanged cytotoxicity, changed cytostatic effects, and/or changed serumhalf-life.

There are a wide variety of expression systems which may be chosen toproduce a polypeptide or cell-targeted protein of the present invention.For example, host organisms for expression of cell-targeted proteins ofthe invention include prokaryotes, such as E. coli and B. subtilis,eukaryotic cells, such as yeast and filamentous fungi (like S.cerevisiae, P. pastoris, A. awamori, and K. lactis), algae (like C.reinhardtii), insect cell lines, mammalian cells (like CHO cells), plantcell lines, and eukaryotic organisms such as transgenic plants (like A.thaliana and N. benthamiana).

Accordingly, the present invention also provides methods for producing aCD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptide and/or cell-targeted protein of the invention according toabove recited methods and using a polynucleotide encoding part or all ofa polypeptide of the invention or a polypeptide component of acell-targeted protein of the invention, an expression vector comprisingat least one polynucleotide of the invention capable of encoding part orall of a polypeptide of the invention when introduced into a host cell,and/or a host cell comprising a polynucleotide or expression vector ofthe invention.

When a polypeptide or protein is expressed using recombinant techniquesin a host cell or cell-free system, it is advantageous to separate (orpurify) the desired polypeptide or protein away from other components,such as host cell factors, in order to obtain preparations that are ofhigh purity or are substantially homogeneous. Purification can beaccomplished by methods well known in the art, such as centrifugationtechniques, extraction techniques, chromatographic and fractionationtechniques (e.g. size separation by gel filtration, charge separation byion-exchange column, hydrophobic interaction chromatography, reversephase chromatography, chromatography on silica or cation-exchange resinssuch as DEAE and the like, chromatofocusing, and Protein A Sepharosechromatography to remove contaminants), and precipitation techniques(e.g. ethanol precipitation or ammonium sulfate precipitation). Anynumber of biochemical purification techniques may be used to increasethe purity of a CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cellde-immunized polypeptide and/or cell-targeted molecule of the presentinvention. In certain embodiments, the polypeptides and cell-targetedmolecules of the invention may optionally be purified in homo-multimericforms (i.e. a protein complex of two or more identical polypeptides orcell-targeted molecules of the invention).

In the Examples below are descriptions of non-limiting examples ofmethods for producing a polypeptide or cell-targeted molecule of theinvention, as well as specific but non-limiting aspects of productionfor exemplary cell-targeted molecules of the present invention.

VIII. Pharmaceutical and Diagnostic Compositions Comprising a T-CellHyper-Immunized and/or B-Cell/CD4+ T-Cell De-Immunized Polypeptide ofthe Present Invention or Cell-Targeted Molecule Comprising the Same

The present invention provides polypeptides and proteins for use, aloneor in combination with one or more additional therapeutic agents, in apharmaceutical composition, for treatment or prophylaxis of conditions,diseases, disorders, or symptoms described in further detail below (e.g.cancers, malignant tumors, non-malignant tumors, growth abnormalities,immune disorders, and microbial infections). The present inventionfurther provides pharmaceutical compositions comprising a polypeptide orcell-targeted molecule of the invention, or a pharmaceuticallyacceptable salt or solvate thereof, according to the invention, togetherwith at least one pharmaceutically acceptable carrier, excipient, orvehicle. In certain embodiments, the pharmaceutical composition of thepresent invention may comprise homo-multimeric and/or hetero-multimericforms of the polypeptides or cell-targeted molecules of the invention.The pharmaceutical compositions will be useful in methods of treating,ameliorating, or preventing a disease, condition, disorder, or symptomdescribed in further detail below. Each such disease, condition,disorder, or symptom is envisioned to be a separate embodiment withrespect to uses of a pharmaceutical composition according to theinvention. The invention further provides pharmaceutical compositionsfor use in at least one method of treatment according to the invention,as described in more detail below.

As used herein, the terms “patient” and “subject” are usedinterchangeably to refer to any organism, commonly vertebrates such ashumans and animals, which presents symptoms, signs, and/or indicationsof at least one disease, disorder, or condition. These terms includemammals such as the non-limiting examples of primates, livestock animals(e.g. cattle, horses, pigs, sheep, goats, etc.), companion animals (e.g.cats, dogs, etc.) and laboratory animals (e.g. mice, rabbits, rats,etc.).

As used herein, “treat,” “treating,” or “treatment” and grammaticalvariants thereof refer to an approach for obtaining beneficial ordesired clinical results. The terms may refer to slowing the onset orrate of development of a condition, disorder or disease, reducing oralleviating symptoms associated with it, generating a complete orpartial regression of the condition, or some combination of any of theabove. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, reduction oralleviation of symptoms, diminishment of extent of disease,stabilization (e.g. not worsening) of state of disease, delay or slowingof disease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treat,” “treating,” or “treatment” can also meanprolonging survival relative to expected survival time if not receivingtreatment. A subject (e.g. a human) in need of treatment may thus be asubject already afflicted with the disease or disorder in question. Theterms “treat,” “treating,” or “treatment” includes inhibition orreduction of an increase in severity of a pathological state or symptomsrelative to the absence of treatment, and is not necessarily meant toimply complete cessation of the relevant disease, disorder, orcondition. With regard to tumors and/or cancers, treatment includesreduction in overall tumor burden and/or individual tumor size.

As used herein, the terms “prevent,” “preventing,” “prevention” andgrammatical variants thereof refer to an approach for preventing thedevelopment of, or altering the pathology of, a condition, disease, ordisorder. Accordingly, “prevention” may refer to prophylactic orpreventive measures. For the purposes of this invention, beneficial ordesired clinical results include, but are not limited to, prevention orslowing of symptoms, progression or development of a disease, whetherdetectable or undetectable. A subject (e.g. a human) in need ofprevention may thus be a subject not yet afflicted with the disease ordisorder in question. The term “prevention” includes slowing the onsetof disease relative to the absence of treatment, and is not necessarilymeant to imply permanent prevention of the relevant disease, disorder orcondition. Thus “preventing” or “prevention” of a condition may incertain contexts refer to reducing the risk of developing the condition,or preventing or delaying the development of symptoms associated withthe condition.

As used herein, an “effective amount” or “therapeutically effectiveamount” is an amount or dose of a composition (e.g. a therapeuticcomposition or agent) that produces at least one desired therapeuticeffect in a subject, such as preventing or treating a target conditionor beneficially alleviating a symptom associated with the condition. Themost desirable therapeutically effective amount is an amount that willproduce a desired efficacy of a particular treatment selected by one ofskill in the art for a given subject in need thereof. This amount willvary depending upon a variety of factors understood by the skilledworker, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type, disease stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, namely by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly (seee.g. Remington: The Science and Practice of Pharmacy (Gennaro A, ed.,Mack Publishing Co., Easton, Pa., U.S., 19th ed., 1995)).

Diagnostic compositions comprise a polypeptide or cell-targeted moleculeof the invention and one or more detection promoting agents. Variousdetection promoting agents are known in the art, such as isotopes, dyes,colorimetric agents, contrast enhancing agents, fluorescent agents,bioluminescent agents, and magnetic agents. These agents may beincorporated into the polypeptide or cell-targeted molecule of theinvention at any position. The incorporation of the agent may be via anamino acid residue(s) of the protein or via some type of linkage knownin the art, including via linkers and/or chelators. The incorporation ofthe agent is in such a way to enable the detection of the presence ofthe diagnostic composition in a screen, assay, diagnostic procedure,and/or imaging technique.

When producing or manufacturing a diagnostic composition of the presentinvention, a cell-targeted molecule of the invention may be directly orindirectly linked to one or more detection promoting agents. There arenumerous detection promoting agents known to the skilled worker whichcan be operably linked to the polypeptides or cell-targeted molecules ofthe invention for information gathering methods, such as for diagnosticand/or prognostic applications to diseases, disorders, or conditions ofan organism (see e.g. Cai W et al., J Nucl Med 48: 304-10 (2007); NayakT, Brechbiel M, Bioconjug Chem 20: 825-41 (2009); Paudyal P et al.,Oncol Rep 22: 115-9 (2009); Qiao J et al., PLoS ONE 6: e18103 (2011);Sano K et al., Breast Cancer Res 14: R61 (2012)). For example, detectionpromoting agents include image enhancing contrast agents, such asfluorescent dyes (e.g. Alexa680, indocyanine green, and Cy5.5), isotopesand radionuclides, such as ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ³²P, ⁵¹Mn, ⁵²mMn, ⁵²Fe,⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁷³Se, ⁷⁵Br, ⁷⁶Br, ⁸²mRb, ⁸³Sr,⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ⁹⁴mTc, ⁹⁴Tc, ⁹⁹mTc, ¹¹⁰In, ¹¹¹In, ¹²⁰I, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I, ¹⁵⁴Gd, ¹⁵⁵Gd, ¹⁵⁶Gd, ¹⁵⁷Gd, ¹⁵⁸Gd, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, and²²³R; paramagnetic ions, such as chromium (III), manganese (II), iron(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III); metals,such as lanthanum (III), gold (III), lead (II), and bismuth (III);ultrasound-contrast enhancing agents, such as liposomes; radiopaqueagents, such as barium, gallium, and thallium compounds. Detectionpromoting agents may be incorporated directly or indirectly by using anintermediary functional group, such as chelators like 2-benzyl DTPA,PAMAM, NOTA, DOTA, TETA, analogs thereof, and functional equivalents ofany of the foregoing (see Leyton J et al., Clin Cancer Res 14: 7488-96(2008)).

There are numerous standard techniques known to the skilled worker forincorporating, affixing, and/or conjugating various detection promotingagents to proteins, especially to immunoglobulins andimmunoglobulin-derived domains (Wu A, Methods 65: 139-47 (2014)).Similarly, there are numerous imaging approaches known to the skilledworker, such as non-invasive in vivo imaging techniques commonly used inthe medical arena, for example: computed tomography imaging (CTscanning), optical imaging (including direct, fluorescent, andbioluminescent imaging), magnetic resonance imaging (MRI), positronemission tomography (PET), single-photon emission computed tomography(SPECT), ultrasound, and x-ray computed tomography imaging (see Kaur Set al., Cancer Lett 315: 97-111 (2012), for review).

IX. Production or Manufacture of a Pharmaceutical and/or DiagnosticComposition Comprising a T-Cell Hyper-Immunized and/or B-Cell/CD4+T-Cell De-Immunized Polypeptide or Cell-Targeted Molecule of the PresentInvention

Pharmaceutically acceptable salts or solvates of any of the polypeptidesand cell-targeted molecules of the invention are likewise within thescope of the present invention.

The term “solvate” in the context of the present invention refers to acomplex of defined stoichiometry formed between a solute (in casu, apolypeptide compound or pharmaceutically acceptable salt thereofaccording to the invention) and a solvent. The solvent in thisconnection may, for example, be water, ethanol or anotherpharmaceutically acceptable, typically small-molecular organic species,such as, but not limited to, acetic acid or lactic acid. When thesolvent in question is water, such a solvate is normally referred to asa hydrate.

Polypeptides and proteins of the present invention, or salts thereof,may be formulated as pharmaceutical compositions prepared for storage oradministration, which typically comprise a therapeutically effectiveamount of a compound of the present invention, or a salt thereof, in apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” includes any of the standard pharmaceuticalcarriers. Pharmaceutically acceptable carriers for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences (Mack Publishing Co. (A. Gennaro,ed., 1985). As used herein, “pharmaceutically acceptable carrier”includes any and all physiologically acceptable, i.e. compatible,solvents, dispersion media, coatings, antimicrobial agents, isotonic,and absorption delaying agents, and the like. Pharmaceuticallyacceptable carriers or diluents include those used in formulationssuitable for oral, rectal, nasal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. Exemplary pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Examples of suitable aqueous and nonaqueous carriers thatmay be employed in the pharmaceutical compositions of the inventioninclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyloleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants. In certain embodiments, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion). Depending onselected route of administration, the protein or other pharmaceuticalcomponent may be coated in a material intended to protect the compoundfrom the action of low pH and other natural inactivating conditions towhich the active protein may encounter when administered to a patient bya particular route of administration.

The formulations of the pharmaceutical compositions of the invention mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. In such form, thecomposition is divided into unit doses containing appropriate quantitiesof the active component. The unit dosage form can be a packagedpreparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. Compositions may be formulated for anysuitable route and means of administration. Subcutaneous or transdermalmodes of administration may be particularly suitable for therapeuticproteins described herein.

The pharmaceutical compositions of the present invention may alsocontain adjuvants such as preservatives, wetting agents, emulsifyingagents and dispersing agents. Preventing the presence of microorganismsmay be ensured both by sterilization procedures, and by the inclusion ofvarious antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. Isotonic agents, suchas sugars, sodium chloride, and the like into the compositions, may alsobe desirable. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as, aluminum monostearate and gelatin.

A pharmaceutical composition of the present invention also optionallyincludes a pharmaceutically acceptable antioxidant. Exemplarypharmaceutically acceptable antioxidants are water soluble antioxidantssuch as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and thelike; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, andthe like.

In another aspect, the present invention provides pharmaceuticalcompositions comprising one or a combination of different polypeptidesand/or cell-targeted molecules of the invention, or an ester, salt oramide of any of the foregoing, and at least one pharmaceuticallyacceptable carrier.

Therapeutic compositions are typically sterile and stable under theconditions of manufacture and storage. The composition may be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier may be a solvent ordispersion medium containing, for example, water, alcohol such asethanol, polyol (e.g. glycerol, propylene glycol, and liquidpolyethylene glycol), or any suitable mixtures. The proper fluidity maybe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by use of surfactants according to formulation chemistry well knownin the art. In certain embodiments, isotonic agents, e.g. sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride may bedesirable in the composition. Prolonged absorption of injectablecompositions may be brought about by including in the composition anagent that delays absorption for example, monostearate salts andgelatin.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of: a sterile diluent such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates; and tonicity adjusting agents such as, e.g., sodium chlorideor dextrose. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide, or buffers with citrate,phosphate, acetate and the like. Such preparations may be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

Sterile injectable solutions may be prepared by incorporating apolypeptide or cell-targeted molecule of the invention in the requiredamount in an appropriate solvent with one or a combination ofingredients described above, as required, followed by sterilizationmicrofiltration. Dispersions may be prepared by incorporating the activecompound into a sterile vehicle that contains a dispersion medium andother ingredients, such as those described above. In the case of sterilepowders for the preparation of sterile injectable solutions, the methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient in addition to any additionaldesired ingredient from a sterile-filtered solution thereof.

When a therapeutically effective amount of a polypeptide orcell-targeted molecule of the invention is designed to be administeredby, e.g. intravenous, cutaneous or subcutaneous injection, the bindingagent will be in the form of a pyrogen-free, parenterally acceptableaqueous solution. Methods for preparing parenterally acceptable proteinsolutions, taking into consideration appropriate pH, isotonicity,stability, and the like, are within the skill in the art. A preferredpharmaceutical composition for intravenous, cutaneous, or subcutaneousinjection will contain, in addition to binding agents, an isotonicvehicle such as sodium chloride injection, Ringer's injection, dextroseinjection, dextrose and sodium chloride injection, lactated Ringer'sinjection, or other vehicle as known in the art. A pharmaceuticalcomposition of the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additives well known tothose of skill in the art.

As described elsewhere herein, a polypeptide or cell-targeted moleculeof the invention may be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art (see e.g. Sustained andControlled Release Drug Delivery Systems (Robinson J, ed., MarcelDekker, Inc., NY, U.S., 1978)).

In certain embodiments, the pharmaceutical composition of the presentinvention may be formulated to ensure a desired distribution in vivo.For example, the blood-brain barrier excludes many large and/orhydrophilic compounds. To target a therapeutic compound or compositionof the invention to a particular in vivo location, they can beformulated, for example, in liposomes which may comprise one or moremoieties that are selectively transported into specific cells or organs,thus enhancing targeted drug delivery. Exemplary targeting moietiesinclude folate or biotin; mannosides; antibodies; surfactant protein Areceptor; p120 catenin and the like.

Pharmaceutical compositions include parenteral formulations designed tobe used as implants or particulate systems. Examples of implants aredepot formulations composed of polymeric or hydrophobic components suchas emulsions, ion exchange resins, and soluble salt solutions. Examplesof particulate systems are microspheres, microparticles, nanocapsules,nanospheres, and nanoparticles (see e.g. Honda M et al., Int JNanomedicine 8: 495-503 (2013); Sharma A et al., Biomed Res Int 2013:960821 (2013); Ramishetti S, Huang L, Ther Deliv 3: 1429-45 (2012)).Controlled release formulations may be prepared using polymers sensitiveto ions, such as, e.g. liposomes, polaxamer 407, and hydroxyapatite.

X. Polynucleotides, Expression Vectors, and Host Cells

Beyond the polypeptides and proteins of the present invention, thepolynucleotides that encode the polypeptides and cell-targeted moleculesof the invention, or functional portions thereof, are also encompassedwithin the scope of the present invention. The term “polynucleotide” isequivalent to the term “nucleic acid,” each of which includes one ormore of: polymers of deoxyribonucleic acids (DNAs), polymers ofribonucleic acids (RNAs), analogs of these DNAs or RNAs generated usingnucleotide analogs, and derivatives, fragments and homologs thereof. Thepolynucleotide of the present invention may be single-, double-, ortriple-stranded. Such polynucleotides are specifically disclosed toinclude all polynucleotides capable of encoding an exemplary protein,for example, taking into account the wobble known to be tolerated in thethird position of RNA codons, yet encoding for the same amino acid as adifferent RNA codon (see Stothard P, Biotechniques 28: 1102-4 (2000)).

In one aspect, the invention provides polynucleotides which encode aCD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptide and/or cell-targeted protein of the invention, or a fragmentor derivative thereof. The polynucleotides may include, e.g., nucleicacid sequence encoding a polypeptide at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99% or more, identical to a polypeptidecomprising one of the amino acid sequences of the protein. The inventionalso includes polynucleotides comprising nucleotide sequences thathybridize under stringent conditions to a polynucleotide which encodesCD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptide and/or cell-targeted protein of the invention, or a fragmentor derivative thereof, or the antisense or complement of any suchsequence.

Derivatives or analogs of the molecules (e.g., CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptidesand/or cell-targeted proteins comprising the same) of the presentinvention include, inter alia, polynucleotide (or polypeptide) moleculeshaving regions that are substantially homologous to the polynucleotides,CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides, or cell-targeted proteins of the present invention, e.g.by at least about 45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity(with a preferred identity of 80-99%) over a polynucleotide orpolypeptide sequence of the same size or when compared to an alignedsequence in which the alignment is done by a computer homology programknown in the art. An exemplary program is the GAP program (WisconsinSequence Analysis Package, Version 8 for UNIX, Genetics Computer Group,University Research Park, Madison, Wis., U.S.) using the defaultsettings, which uses the algorithm of Smith T, Waterman M, Adv. Appl.Math. 2: 482-9 (1981). Also included are polynucleotides capable ofhybridizing to the complement of a sequence encoding the cell-targetedproteins of the invention under stringent conditions (see e.g. Ausubel Fet al., Current Protocols in Molecular Biology (John Wiley & Sons, NewYork, N.Y., U.S., 1993)), and below. Stringent conditions are known tothose skilled in the art and may be found, e.g., in Current Protocols inMolecular Biology (John Wiley & Sons, NY, U.S., Ch. Sec. 6.3.1-6.3.6(1989)).

The present invention further provides expression vectors that comprisethe polynucleotides within the scope of the present invention. Thepolynucleotides capable of encoding the CD8+ T-cell hyper-immunizedand/or B-cell/CD4+ T-cell de-immunized polypeptides and/or cell-targetedproteins of the invention may be inserted into known vectors, includingbacterial plasmids, viral vectors and phage vectors, using material andmethods well known in the art to produce expression vectors. Suchexpression vectors will include the polynucleotides necessary to supportproduction of contemplated CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptides and/or cell-targetedproteins of the invention within any host cell of choice or cell-freeexpression systems (e.g. pTxb1 and pIVEX2.3). The specificpolynucleotides comprising expression vectors for use with specifictypes of host cells or cell-free expression systems are well known toone of ordinary skill in the art, can be determined using routineexperimentation, or may be purchased.

The term “expression vector,” as used herein, refers to apolynucleotide, linear or circular, comprising one or more expressionunits. The term “expression unit” denotes a polynucleotide segmentencoding a polypeptide of interest and capable of providing expressionof the nucleic acid segment in a host cell. An expression unit typicallycomprises a transcription promoter, an open reading frame encoding thepolypeptide of interest, and a transcription terminator, all in operableconfiguration. An expression vector contains one or more expressionunits. Thus, in the context of the present invention, an expressionvector encoding a CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cellde-immunized polypeptide and/or protein comprising a single polypeptidechain (e.g. a scFv genetically recombined with a CD8+ T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized Shiga toxineffector region) includes at least an expression unit for the singlepolypeptide chain, whereas a protein comprising, e.g. two or morepolypeptide chains (e.g. one chain comprising a V_(L) domain and asecond chain comprising a V_(H) domain linked to a toxin effectorregion) includes at least two expression units, one for each of the twopolypeptide chains of the protein. For expression of multi-chaincell-targeted proteins of the invention, an expression unit for eachpolypeptide chain may also be separately contained on differentexpression vectors (e.g. expression may be achieved with a single hostcell into which expression vectors for each polypeptide chain has beenintroduced).

Expression vectors capable of directing transient or stable expressionof polypeptides and proteins are well known in the art. The expressionvectors generally include, but are not limited to, one or more of thefollowing: a heterologous signal sequence or peptide, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence, each of which is well known inthe art. Optional regulatory control sequences, integration sequences,and useful markers that can be employed are known in the art.

The term “host cell” refers to a cell which can support the replicationor expression of the expression vector. Host cells may be prokaryoticcells, such as E. coli or eukaryotic cells (e.g. yeast, insect,amphibian, bird, or mammalian cells). Creation and isolation of hostcell lines comprising a polynucleotide of the invention or capable ofproducing a polypeptide and/or cell-targeted protein of the inventioncan be accomplished using standard techniques known in the art.

CD8+ T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptides and/or proteins within the scope of the present inventionmay be variants or derivatives of the polypeptides and proteinsdescribed herein that are produced by modifying the polynucleotideencoding a polypeptide and/or protein by altering one or more aminoacids or deleting or inserting one or more amino acids that may renderit more suitable to achieve desired properties, such as more optimalexpression by a host cell.

XI. Delivery Devices and Kits

In certain embodiments, the invention relates to a device comprising oneor more compositions of matter of the invention, such as apharmaceutical composition, for delivery to a subject in need thereof.Thus, a delivery device comprising one or more compounds of theinvention can be used to administer to a patient a composition of matterof the invention by various delivery methods, including: intravenous,subcutaneous, intramuscular or intraperitoneal injection; oraladministration; transdermal administration; pulmonary or transmucosaladministration; administration by implant, osmotic pump, cartridge ormicro pump; or by other means recognized by a person of skill in theart.

Also within the scope of the invention are kits comprising at least onecomposition of matter of the invention, and optionally, packaging andinstructions for use. Kits may be useful for drug administration and/ordiagnostic information gathering. A kit of the invention may optionallycomprise at least one additional reagent (e.g., standards, markers andthe like). Kits typically include a label indicating the intended use ofthe contents of the kit. The kit may further comprise reagents and othertools for detecting a cell type (e.g. tumor cell) in a sample or in asubject, or for diagnosing whether a patient belongs to a group thatresponds to a therapeutic strategy which makes use of a compound,composition or related method of the invention as described herein.

XII. Methods of Generating T-Cell Hyper-Immunized and/or B-Cell/CD4+T-Cell De-Immunized Polypeptides of the Present Invention

The present invention provides methods of creating T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptides ofthe present invention by modifying polypeptides already capable ofintracellularly routing to a cytosol, ER, or lysosome of a cell from anendosomal compartment of the cell; the method comprising the step ofadding a heterologous T-cell epitope to the polypeptide. In certainfurther methods of the present invention, the heterologous T-cellepitope is embedded or inserted within a polypeptide capable ofintracellularly routing to a cytosol, ER, or lysosome of a cell from anendosomal compartment of the cell.

In certain embodiments of the methods of the present invention, a CD8+T-cell hyper-immunized and/or B-cell/CD4+ T-cell de-immunizedpolypeptide of the present invention is created by modifying apolypeptide already capable of intracellularly routing to a cytosol, ER,or lysosome of a cell from an endosomal compartment of the cell; themethod comprising the step of adding a heterologous T-cell epitope tothe polypeptide. In certain further methods of the present invention,the heterologous T-cell epitope is embedded or inserted within apolypeptide capable of intracellularly routing to a cytosol, ER, orlysosome of a cell from an endosomal compartment of the cell.

In certain embodiments of the methods of the present invention, apolypeptide already capable of intracellularly routing to a cytosol, ER,or lysosome of a cell from an endosomal compartment of the cell iscreated into a T-cell hyper-immunized polypeptide of the presentinvention; the method comprising the step of adding a heterologousT-cell epitope to the polypeptide. In certain further embodiments of themethods of the present invention, a polypeptide already capable ofintracellularly routing to a cytosol, ER, or lysosome of a cell from anendosomal compartment of the cell is created into a CD8+ T-cellhyper-immunized polypeptide of the present invention; the methodcomprising the step of adding a heterologous T-cell epitope to thepolypeptide. In certain further methods of the present invention, theheterologous T-cell epitope is embedded or inserted within a polypeptidecapable of intracellularly routing to a cytosol, ER, or lysosome of acell from an endosomal compartment of the cell.

In certain embodiments of the methods of the present invention, apolypeptide capable of delivering a T-cell epitope for presentation by aMHC class I molecule is created; the method comprising the step ofadding a heterologous T-cell epitope to a polypeptide capable ofintracellular delivery of the T-cell epitope from an endosomalcompartment of a cell to a proteasome of the cell. In certain furthermethods of the present invention, the heterologous T-cell epitope isembedded or inserted within a polypeptide capable of intracellularlyrouting to a cytosol, ER, or lysosome of a cell from an endosomalcompartment of the cell.

In certain embodiments of the methods of the present invention, a T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized polypeptide iscreated; the method comprising the step of inserting or embedding aheterologous T-cell epitope into an endogenous B-cell epitope region ofa polypeptide already capable of intracellularly routing to a cytosol,ER, or lysosome of a cell from an endosomal compartment of the cell.

In certain embodiments of the methods of the present invention, a CD8+T-cell hyper-immunized and B-cell/CD4+ T-cell de-immunized polypeptideof the present invention is created; the method comprising the step ofembedding or inserting a heterologous T-cell epitope into an endogenousB-cell epitope region of a polypeptide already capable ofintracellularly routing to a cytosol, ER, or lysosome of a cell from anendosomal compartment of the cell.

In certain embodiments of the methods of the present invention, apolypeptide already capable of intracellularly routing to a cytosol, ER,or lysosome of a cell from an endosomal compartment of the cell iscreated into a T-cell hyper-immunized and/or B-cell/CD4+ T-cellde-immunized polypeptide of the present invention; the method comprisingthe step of embedding or inserting a heterologous T-cell epitope into anendogenous B-cell epitope region of the polypeptide. In certain furtherembodiments of the methods of the present invention, a polypeptidealready capable of intracellularly routing to a cytosol, ER, or lysosomeof a cell from an endosomal compartment of the cell is created into aCD8+ T-cell hyper-immunized polypeptide of the present invention; themethod comprising the step of embedding or inserting a heterologousT-cell epitope into an endogenous B-cell epitope region of thepolypeptide.

In certain embodiments of the methods of the present invention, ade-immunized polypeptide capable of delivering a T-cell epitope forpresentation by a MHC class I molecule is created; the method comprisingthe step of embedding or inserting a heterologous T-cell epitope into anendogenous B-cell epitope region of a polypeptide capable ofintracellular delivery of the T-cell epitope from an endosomalcompartment of a cell to a proteasome of the cell.

In certain embodiments of the methods of the present invention, ade-immunized polypeptide is created which has reduced B-cellimmunogenicity when administered to a chordate. In certain embodimentsof the methods of the present invention, is a method for reducing B-cellimmunogenicity in a polypeptide, the method comprising the step ofdisrupting a B-cell epitope region within a polypeptide with one or moreamino acid residue(s) comprised by a heterologous T-cell epitope addedto the polypeptide. In certain further embodiments, the disrupting stepfurther comprises creating one or more amino acid substitutions in theB-cell epitope region. In certain further embodiments, the disruptingstep further comprises creating one or more amino acid insertions in theB-cell epitope region.

Certain embodiments of the methods of the present invention are methodsfor reducing B-cell immunogenicity in a polypeptide while simultaneouslyincreasing CD8+ T-cell immunogenicity after administration to achordate, the methods comprising the step of disrupting a B-cell epitoperegion within a polypeptide with one or more amino acid residue(s)comprised by a heterologous CD8+ T-cell epitope added to thepolypeptide. In certain further embodiments, the disrupting step furthercomprises creating one or more amino acid substitutions in the B-cellepitope region. In certain further embodiments, the disrupting stepfurther comprises creating one or more amino acid insertions in theB-cell epitope region.

Certain embodiments of the methods of the present invention are methodsfor reducing B-cell immunogenicity in a polypeptide while simultaneouslyincreasing CD8+ T-cell immunogenicity after administration to achordate, the methods comprising the steps of: 1) identifying a B-cellepitope in a polypeptide; and 2) disrupting the identified B-cellepitope with one or more amino acid residue(s) comprised by aheterologous CD8+ T-cell epitope added to the polypeptide. In certainfurther embodiments, the disrupting step further comprises the creationof one or more amino acid substitutions in the B-cell epitope region. Incertain further embodiments, the disrupting step further comprisescreating one or more amino acid insertions in the B-cell epitope region.

Certain embodiments of the methods of the present invention are methodsfor reducing B-cell immunogenicity in a polypeptide while simultaneouslyincreasing CD8+ T-cell immunogenicity after administration to achordate, the methods comprising the steps of: 1) identifying a B-cellepitope in a polypeptide; and 2) disrupting the identified B-cellepitope with one or more amino acid residue(s) comprised by aheterologous CD8+ T-cell epitope added to the polypeptide. In certainfurther embodiments, the disrupting step further comprises the creationof one or more amino acid substitutions in the B-cell epitope region. Incertain further embodiments, the disrupting step further comprisescreating one or more amino acid insertions in the B-cell epitope region.

In certain embodiments of the methods of the present invention, a CD4+T-cell de-immunized polypeptide is created which has reduced CD4+ T-cellimmunogenicity when administered to a chordate. In certain embodimentsof the methods of the present invention, is a method for reducing CD4+T-cell immunogenicity in a polypeptide, the method comprising the stepof disrupting a CD4+ T-cell epitope region within a polypeptide with oneor more amino acid residue(s) comprised by a heterologous CD8+ T-cellepitope added to the polypeptide. In certain further embodiments, thedisrupting step further comprises creating one or more amino acidsubstitutions in the B-cell epitope region. In certain furtherembodiments, the disrupting step further comprises creating one or moreamino acid insertions in the CD4+ T-cell epitope region.

Certain embodiments of the methods of the present invention are methodsfor reducing CD4+ T-cell immunogenicity in a polypeptide whilesimultaneously increasing CD8+ T-cell immunogenicity afteradministration to a chordate, the methods comprising the step ofdisrupting a CD4+ T-cell epitope region within a polypeptide with one ormore amino acid residue(s) comprised by a heterologous CD8+ T-cellepitope added to the polypeptide. In certain further embodiments, thedisrupting step further comprises creating one or more amino acidsubstitutions in the CD4+ T-cell epitope region. In certain furtherembodiments, the disrupting step further comprises creating one or moreamino acid insertions in the CD4+ T-cell epitope region.

Certain embodiments of the methods of the present invention are methodsfor reducing CD4+ T-cell immunogenicity in a polypeptide whilesimultaneously increasing CD8+ T-cell immunogenicity afteradministration to a chordate, the methods comprising the steps of: 1)identifying a CD4+ T-cell epitope in a polypeptide; and 2) disruptingthe identified CD4+ T-cell epitope with one or more amino acidresidue(s) comprised by a heterologous CD8+ T-cell epitope added to thepolypeptide. In certain further embodiments, the disrupting step furthercomprises the creation of one or more amino acid substitutions in theCD4+ T-cell epitope region. In certain further embodiments, thedisrupting step further comprises creating one or more amino acidinsertions in the CD4+ T-cell epitope region.

Certain embodiments of the methods of the present invention are methodsfor reducing CD4+ T-cell immunogenicity in a polypeptide whilesimultaneously increasing CD8+ T-cell immunogenicity afteradministration to a chordate, the methods comprising the steps of: 1)identifying a CD4+ T-cell epitope in a polypeptide; and 2) disruptingthe identified CD4+ T-cell epitope with one or more amino acidresidue(s) comprised by a heterologous CD8+ T-cell epitope added to thepolypeptide. In certain further embodiments, the disrupting step furthercomprises the creation of one or more amino acid substitutions in theCD4+ T-cell epitope region. In certain further embodiments, thedisrupting step further comprises creating one or more amino acidinsertions in the CD4+ T-cell epitope region.

XIII. Methods for Using a T-Cell Hyper-Immunized and/or B-Cell/CD4+T-Cell De-Immunized Polypeptide of the Present Invention, Cell-TargetedMolecule Comprising the Same, or Pharmaceutical and/or DiagnosticComposition Thereof

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the prevention and/or treatment of diseases, disorders, andconditions, such as certain cancers, tumors, growth abnormalities,immune disorders, or further pathological conditions mentioned herein.Accordingly, the present invention provides methods of using thepolypeptides, cell-targeted molecules, and pharmaceutical compositionsof the present invention for the delivering of T-cell epitopes to theMHC class I presentation pathway of target cells, targeted killing ofcells, for delivering additional exogenous materials into targetedcells, for labeling of the interiors of targeted cells, for collectingdiagnostic information, and for treating diseases, disorders, andconditions as described herein.

Already cytotoxic molecules, such as e.g. potential therapeuticscomprising cytotoxic toxin region polypeptides, may be engineered to bemore cytotoxic and/or to have redundant, backup cytotoxicities operatingvia completely different mechanisms. These multiple cytotoxic mechanismsmay complement each other (such as by providing both two mechanisms ofcell killing, direct and indirect, as well as mechanisms ofimmuno-stimulation to the local area), redundantly backup each other(such as by providing direct cell killing in the absence of the other),and/or protect against developed resistance (by limiting resistance tothe less probable situation of the malignant or infected cell blockingtwo different mechanisms simultaneously).

In addition, parental cytotoxic molecules which rely on toxin effectorand/or enzymatic regions for cytotoxicity may be engineered by mutatingthe parental molecule to be enzymatically inactive but to be cytotoxicvia T-cell epitope delivery to the MHC class I system of a target celland subsequent presentation to the surface of the target cell. Thisapproach removes one cytotoxic mechanism while adding another and addsthe capability of immuno-stimulation to the local area of the targetcell by T-cell epitope presentation. Furthermore, parental cytotoxicmolecules which rely on enzymatic regions for cytotoxicity may beengineered to be cytotoxic only via T-cell epitope delivery to the MHCclass I system by embedding a T-cell epitope in the enzymatic domain ofthe parental molecule such that the enzymatic activity is reduced oreliminated. This allows for the one-step modification ofenzymatically-cytotoxic molecules, which have the ability once in anendosomal compartment to route to the cytosol and/or ER, intoenzymatically inactive, cytotoxic molecules which rely on T-cell epitopedelivery to the MHC class I system of a target cell and subsequentpresentation on the surface of the target cell for cytotoxicity. Any ofthe polypeptides of the invention can be engineered into cell-targetedcytotoxic molecules with potential as therapeutics by the linking of avariety of cell-targeting binding regions which target specificcell-type(s) within a mixture of two or more cell types, such as, e.g.,within an organism.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions, and/or methods that havecertain advantages compared to the agents, compositions, and/or methodsthat are currently known in the art. Accordingly, the present inventionprovides methods of using polypeptides and proteins with characterizedby polypeptide sequences and pharmaceutical compositions thereof. Forexample, any of the polypeptide sequences in SEQ ID NOs: 1-60 may bespecifically utilized as a component of the cell-targeted molecules usedin the following methods.

The present invention provides methods of killing a cell comprising thestep of contacting the cell, either in vitro or in vivo, with apolypeptide, protein, or pharmaceutical composition of the presentinvention. The polypeptides, proteins, and pharmaceutical compositionsof the present invention can be used to kill a specific cell type uponcontacting a cell or cells with one of the claimed compositions ofmatter. In certain embodiments, a cytotoxic polypeptide, protein, orpharmaceutical composition of the present invention can be used to killspecific cell types in a mixture of different cell types, such asmixtures comprising cancer cells, infected cells, and/or hematologicalcells. In certain embodiments, a cytotoxic polypeptide, protein, orpharmaceutical composition of the present invention can be used to killcancer cells in a mixture of different cell types. In certainembodiments, a cytotoxic polypeptide, protein, or pharmaceuticalcomposition of the present invention can be used to kill specific celltypes in a mixture of different cell types, such as pre-transplantationtissues. In certain embodiments, a polypeptide, protein, orpharmaceutical composition of the present invention can be used to killspecific cell types in a mixture of cell types, such aspre-administration tissue material for therapeutic purposes. In certainembodiments, a polypeptide, protein, or pharmaceutical composition ofthe present invention can be used to selectively kill cells infected byviruses or microorganisms, or otherwise selectively kill cellsexpressing a particular extracellular target biomolecule, such as a cellsurface biomolecule. The polypeptides, proteins, and pharmaceuticalcompositions of the present invention have varied applications,including, e.g., uses in depleting unwanted cell types from tissueseither in vitro or in vivo, uses in modulating immune responses to treatgraft-versus-host disease, uses as antiviral agents, uses asanti-parasitic agents, and uses in purging transplantation tissues ofunwanted cell types.

In certain embodiments, a cytotoxic polypeptide, protein, orpharmaceutical composition of the present invention, alone or incombination with other compounds or pharmaceutical compositions, canshow potent cell-kill activity when administered to a population ofcells, in vitro or in vivo in a subject such as in a patient in need oftreatment. By targeting the delivery of enzymatically active toxinregions and T-cell epitopes using high-affinity binding regions tospecific cell types, this potent cell-kill activity can be restricted tospecifically and selectively kill certain cell types within an organism,such as certain cancer cells, neoplastic cells, malignant cells,non-malignant tumor cells, or infected cells.

The present invention provides a method of killing a cell in a patientin need thereof, the method comprising the step of administering to thepatient at least one cytotoxic polypeptide or protein of the presentinvention, or a pharmaceutical composition thereof.

Certain embodiments of the cytotoxic polypeptide, protein, orpharmaceutical compositions thereof can be used to kill a cancer cell ina patient by targeting an extracellular biomolecule found physicallycoupled with a cancer or tumor cell. The terms “cancer cell” or“cancerous cell” refers to various neoplastic cells which grow anddivide in an abnormally accelerated fashion and will be clear to theskilled person. The term “tumor cell” includes both malignant andnon-malignant cells. Generally, cancers and/or tumors can be defined asdiseases, disorders, or conditions that are amenable to treatment and/orprevention. The cancers and tumors (either malignant or non-malignant)which are comprised of cancer cells and/or tumor cells which may benefitfrom methods and compositions of the invention will be clear to theskilled person. Neoplastic cells are often associated with one or moreof the following: unregulated growth, lack of differentiation, localtissue invasion, angiogenesis, and metastasis.

Certain embodiments of the cytotoxic polypeptide or cell-targetedmolecule of the present invention, or pharmaceutical compositionsthereof, can be used to kill an immune cell (whether healthy ormalignant) in a patient by targeting an extracellular biomolecule foundphysically coupled with an immune cell.

Certain embodiments of the cytotoxic polypeptide or cell-targetedmolecule of the present invention, or pharmaceutical compositionsthereof, can be used to kill an infected cell in a patient by targetingan extracellular biomolecule found physically coupled with an infectedcell.

It is within the scope of the present invention to utilize thecell-targeted molecule of the present invention or pharmaceuticalcomposition thereof for the purposes of purging patient cell populations(e.g. bone marrow) of malignant, neoplastic, or otherwise unwantedT-cells and/or B-cells and then reinfusing the T-cell and/or B-cellsdepleted material into the patient (see e.g. van Heeckeren W et al., BrJ Haematol 132: 42-55 (2006); (see e.g. Alpdogan O, van den Brink M,Semin Oncol 39: 629-42 (2012)).

It is within the scope of the present invention to utilize thecell-targeted molecule of the present invention or pharmaceuticalcomposition thereof for the purposes of ex vivo depletion of T cellsand/or B-cells from isolated cell populations removed from a patient. Inone non-limiting example, the cell-targeted molecule of the inventioncan be used in a method for prophylaxis of organ and/or tissuetransplant rejection wherein the donor organ or tissue is perfused priorto transplant with a cytotoxic, cell-targeted molecule of the inventionor a pharmaceutical composition thereof in order to purge the organ ofdonor T-cells and/or B-cells (see e.g. Alpdogan O, van den Brink M,Semin Oncol 39: 629-42 (2012)).

It is also within the scope of the present invention to utilize thecell-targeted molecule of the invention or pharmaceutical compositionthereof for the purposes of depleting T-cells and/or B-cells from adonor cell population as a prophylaxis against graft-versus-hostdisease, and induction of tolerance, in a patient to undergo a bonemarrow and or stem cell transplant (see e.g. van Heeckeren W et al., BrJ Haematol 132: 42-55 (2006); (see e.g. Alpdogan O, van den Brink M,Semin Oncol 39: 629-42 (2012)).

Certain embodiments of the cytotoxic polypeptide or cell-targetedmolecule of the invention, or pharmaceutical compositions thereof, canbe used to kill an infected cell in a patient by targeting anextracellular biomolecule found physically coupled with an infectedcell.

Certain embodiments of the cell-targeted molecules of the presentinvention, or pharmaceutical compositions thereof, can be used to “seed”a locus within an organism with non-self, T-cell epitope-peptidepresenting cells in order to activate the immune system to police thelocus. In certain further embodiments of this “seeding” method of thepresent invention, the locus is a tumor mass or infected tissue site. Inpreferred embodiments of this “seeding” method of the present invention,the non-self, T-cell epitope-peptide is selected from the groupconsisting of: peptides not already presented by the target cells of thecell-targeted molecule, peptides not present within any proteinexpressed by the target cell, peptides not present within the proteomeof the target cell, peptides not present in the extracellularmicroenvironment of the site to be seeded, and peptides not present inthe tumor mass or infect tissue site to be targeted.

This “seeding” method functions to label one or more target cells withina chordate with one or more MHC class I presented T-cell epitopes forrecognition by effector T-cells and activation of downstream immuneresponses. By exploiting the cell internalizing, intracellularlyrouting, and T-cell epitope delivering functions of the cell-targetedmolecules of the invention, the target cells which display the deliveredT-cell epitope are harnessed to induce recognition of the presentingtarget cell by host T-cells and induction of further immune responsesincluding target cell killing by CTLs. This “seeding” method of using acell-targeted molecule of the present invention can provide a temporaryvaccination-effect by inducing adaptive immune responses to attack thecells within the seeded microenvironment, such as, e.g. a tumor mass orinfected tissue site, whether presenting a cell-targetedmolecule-delivered T-cell epitope(s) or not. This “seeding” method mayalso induce the breaking of immuno-tolerance to a target cellpopulation, a tumor mass, and/or infected tissue site within anorganism.

Additionally, the present invention provides a method of treating adisease, disorder, or condition in a patient comprising the step ofadministering to a patient in need thereof a therapeutically effectiveamount of at least one of the cytotoxic polypeptide or cell-targetedmolecule of the present invention, or a pharmaceutical compositionthereof. Contemplated diseases, disorders, and conditions that can betreated using this method include cancers, malignant tumors,non-malignant tumors, growth abnormalities, immune disorders, andmicrobial infections. Administration of a “therapeutically effectivedosage” of a compound of the invention can result in a decrease inseverity of disease symptoms, an increase in frequency and duration ofdisease symptom-free periods, or a prevention of impairment ordisability due to the disease affliction.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific patientunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy, and may depend on such factors as weight, diet,concurrent medication and other factors, well known to those skilled inthe medical arts. The dosage sizes and dosing regimen most appropriatefor human use may be guided by the results obtained by the presentinvention, and may be confirmed in properly designed clinical trials. Aneffective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

An acceptable route of administration may refer to any administrationpathway known in the art, including but not limited to aerosol, enteral,nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal(e.g. topical administration of a cream, gel or ointment, or by means ofa transdermal patch). “Parenteral administration” is typicallyassociated with injection at or in communication with the intended siteof action, including infraorbital, infusion, intraarterial,intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal administration.

For administration of a pharmaceutical composition of the presentinvention, the dosage range will generally be from about 0.0001 to 100milligrams per kilogram (mg/kg), and more, usually 0.01 to 5 mg/kg, ofthe host body weight. Exemplary dosages may be 0.25 mg/kg body weight, 1mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. An exemplary treatmentregime is a once or twice daily administration, or a once or twiceweekly administration, once every two weeks, once every three weeks,once every four weeks, once a month, once every two or three months oronce every three to 6 months. Dosages may be selected and readjusted bythe skilled health care professional as required to maximize therapeuticbenefit for a particular patient.

Pharmaceutical compositions of the present invention will typically beadministered to the same patient on multiple occasions. Intervalsbetween single dosages can be, for example, 2-5 days, weekly, monthly,every two or three months, every six months, or yearly. Intervalsbetween administrations can also be irregular, based on regulating bloodlevels or other markers in the subject or patient. Dosage regimens for acompound of the invention include intravenous administration of 1 mg/kgbody weight or 3 mg/kg body weight with the compound administered everytwo to four weeks for six dosages, then every three months at 3 mg/kgbody weight or 1 mg/kg body weight.

A pharmaceutical composition of the present invention may beadministered via one or more routes of administration, using one or moreof a variety of methods known in the art. As will be appreciated by theskilled worker, the route and/or mode of administration will varydepending upon the desired results. Routes of administration forpolypeptides, proteins, and pharmaceutical compositions of the inventioninclude, e.g. intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal, or other parenteral routes of administration, forexample by injection or infusion. In other embodiments, a polypeptide,protein, or pharmaceutical composition of the invention may beadministered by a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually, or topically.

Therapeutic polypeptides, proteins, or pharmaceutical compositions ofthe present invention may be administered with one or more of a varietyof medical devices known in the art. For example, in one embodiment, apharmaceutical composition of the invention may be administered with aneedleless hypodermic injection device. Examples of well-known implantsand modules useful in the present invention are in the art, includinge.g., implantable micro-infusion pumps for controlled rate delivery;devices for administering through the skin; infusion pumps for deliveryat a precise infusion rate; variable flow implantable infusion devicesfor continuous drug delivery; and osmotic drug delivery systems. Theseand other such implants, delivery systems, and modules are known tothose skilled in the art.

A polypeptide, protein, or pharmaceutical composition of the presentinvention may be administered alone or in combination with one or moreother therapeutic or diagnostic agents. A combination therapy mayinclude a cytotoxic, cell-targeted molecule of the invention orpharmaceutical composition thereof combined with at least one othertherapeutic agent selected based on the particular patient, disease orcondition to be treated. Examples of other such agents include, interalia, a cytotoxic, anti-cancer or chemotherapeutic agent, ananti-inflammatory or anti-proliferative agent, an antimicrobial orantiviral agent, growth factors, cytokines, an analgesic, atherapeutically active small molecule or polypeptide, a single chainantibody, a classical antibody or fragment thereof, or a nucleic acidmolecule which modulates one or more signaling pathways, and similarmodulating therapeutics which may complement or otherwise be beneficialin a therapeutic or prophylactic treatment regimen.

Treatment of a patient with a polypeptide, protein, or pharmaceuticalcomposition of the present invention preferably leads to cell death oftargeted cells and/or the inhibition of growth of targeted cells. Assuch, cytotoxic, cell-targeted molecules of the present invention, andpharmaceutical compositions comprising them, will be useful in methodsfor treating a variety of pathological disorders in which killing ordepleting target cells may be beneficial, such as, inter alia, cancer,tumors, other growth abnormalities, immune disorders, and infectedcells. The present invention provides methods for suppressing cellproliferation, and treating cell disorders, including neoplasia,overactive B-cells, and overactive T-cells.

In certain embodiments, polypeptides, proteins, and pharmaceuticalcompositions of the present invention can be used to treat or preventcancers, tumors (malignant and non-malignant), growth abnormalities,immune disorders, and microbial infections. In a further aspect, theabove ex vivo method can be combined with the above in vivo method toprovide methods of treating or preventing rejection in bone marrowtransplant recipients, and for achieving immunological tolerance.

In certain embodiments, the present invention provides methods fortreating malignancies or neoplasms and other blood cell associatedcancers in a mammalian subject, such as a human, the method comprisingthe step of administering to a subject in need thereof a therapeuticallyeffective amount of a cytotoxic protein or pharmaceutical composition ofthe invention.

The cytotoxic polypeptides, proteins, and pharmaceutical compositions ofthe present invention have varied applications, including, e.g., uses inremoving unwanted T-cells, uses in modulating immune responses to treatgraft-versus-host disease, uses as antiviral agents, uses asantimicrobial agents, and uses in purging transplantation tissues ofunwanted cell types. The cytotoxic polypeptides, proteins, andpharmaceutical compositions of the present invention are commonlyanti-neoplastic agents—meaning they are capable of treating and/orpreventing the development, maturation, or spread of neoplastic ormalignant cells by inhibiting the growth and/or causing the death ofcancer or tumor cells.

In certain embodiments, a polypeptide, protein, or pharmaceuticalcomposition of the present invention is used to treat a B-cell-, plasmacell- or antibody-mediated disease or disorder, such as for exampleleukemia, lymphoma, myeloma, Human Immunodeficiency Virus-relateddiseases, amyloidosis, hemolytic uremic syndrome, polyarteritis, septicshock, Crohn's Disease, rheumatoid arthritis, ankylosing spondylitis,psoriatic arthritis, ulcerative colitis, psoriasis, asthma, Sjorgren'ssyndrome, graft-versus-host disease, graft rejection, diabetes,vasculitis, scleroderma, and systemic lupus erythematosus.

In another aspect, certain embodiments of the polypeptides, proteins,and pharmaceutical compositions of the present invention areantimicrobial agents—meaning they are capable of treating and/orpreventing the acquisition, development, or consequences ofmicrobiological pathogenic infections, such as caused by viruses,bacteria, fungi, prions, or protozoans.

It is within the scope of the present invention to provide a prophylaxisor treatment for diseases or conditions mediated by T-cells or B-cellsby administering the cytotoxic protein or the invention, or apharmaceutical composition thereof, to a patient for the purpose ofkilling T-cells or B-cells in the patient. This usage is compatible withpreparing or conditioning a patient for bone marrow transplantation,stem cell transplantation, tissue transplantation, or organtransplantation, regardless of the source of the transplanted material,e.g. human or non-human sources.

It is within the scope of the present invention to provide a bone marrowrecipient for prophylaxis or treatment of host-versus-graft disease viathe targeted cell-killing of host T-cells using a cytotoxic polypeptide,protein, or pharmaceutical composition of the present invention.

The cytotoxic polypeptides, proteins, and pharmaceutical compositions ofthe present invention can be utilized in a method of treating cancercomprising administering to a patient, in need thereof, atherapeutically effective amount of a cytotoxic polypeptide, protein, orpharmaceutical composition of the present invention. In certainembodiments of the methods of the present invention, the cancer beingtreated is selected from the group consisting of: bone cancer (such asmultiple myeloma or Ewing's sarcoma), breast cancer, central/peripheralnervous system cancer (such as brain cancer, neurofibromatosis, orglioblastoma), gastrointestinal cancer (such as stomach cancer orcolorectal cancer), germ cell cancer (such as ovarian cancers andtesticular cancers, glandular cancer (such as pancreatic cancer,parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroidcancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer,or pharyngeal cancer), hematological cancers (such as leukemia,lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancerand bladder cancer), liver cancer, lung/pleura cancer (such asmesothelioma, small cell lung carcinoma, or non-small cell lungcarcinoma), prostate cancer, sarcoma (such as angiosarcoma,fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma), skin cancer (suchas basal cell carcinoma, squamous cell carcinoma, or melanoma), anduterine cancer.

The polypeptides, proteins, and pharmaceutical compositions of thepresent invention can be utilized in a method of treating an immunedisorder comprising administering to a patient, in need thereof, atherapeutically effective amount of the cytotoxic protein or apharmaceutical composition of the present invention. In certainembodiments of the methods of the present invention, the immune disorderis related to an inflammation associated with a disease selected fromthe group consisting of: amyloidosis, ankylosing spondylitis, asthma,Crohn's disease, diabetes, graft rejection, graft-versus-host disease,Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-relateddiseases, lupus erythematosus, multiple sclerosis, polyarteritis,psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma,septic shock, Sjorgren's syndrome, ulcerative colitis, and vasculitis.

Among certain embodiments of the present invention is using thepolypeptide or cell-targeted molecule of the present invention as acomponent of a pharmaceutical composition or medicament for thetreatment or prevention of a cancer, tumor, other growth abnormality,immune disorder, and/or microbial infection. For example, immunedisorders presenting on the skin of a patient may be treated with such amedicament in efforts to reduce inflammation. In another example, skintumors may be treated with such a medicament in efforts to reduce tumorsize or eliminate the tumor completely.

Certain cytotoxic polypeptides, proteins, and pharmaceuticalcompositions of the present invention may be used in molecularneurosurgery applications such as immunolesioning and neuronal tracing(see, Wiley R, Lappi D, Adv Drug Deliv Rev 55: 1043-54 (2003), forreview). For example, the targeting domain may be selected or derivedfrom various ligands, such as neurotransmitters and neuropeptides, whichtarget specific neuronal cell types by binding neuronal surfacereceptors, such as a neuronal circuit specific G-protein coupledreceptor. Similarly, the targeting domain may be selected from orderived from antibodies that bind neuronal surface receptors. Becausecertain toxins robustly direct their own retrograde axonal transport,certain cytotoxic, cell-targeted molecules of the invention may be usedto kill a neuron(s) which expresses the extracellular target at a siteof cytotoxic protein injection distant from the cell body (seeLlewellyn-Smith I et al., J Neurosci Methods 103: 83-90 (2000)). Theseneuronal cell type specific targeting cytotoxic polypeptides andproteins have uses in neuroscience research, such as for elucidatingmechanisms of sensations (see e.g. Mishra S, Hoon M, Science 340: 968-71(2013), and creating model systems of neurodegenerative diseases, suchas Parkinson's and Alzheimer's (see e.g. Hamlin A et al., PLoS Onee53472 (2013)).

Among certain embodiment of the present invention is a method of using apolypeptide, protein, pharmaceutical composition, and/or diagnosticcomposition of the present invention to label or detect the interiors ofneoplastic cells and/or immune cell types. Based on the ability ofcertain polypeptides, proteins, and pharmaceutical compositions of theinvention to enter specific cell types and route within cells viaretrograde intracellular transport, the interior compartments ofspecific cell types are labeled for detection. This can be performed oncells in situ within a patient or on cells and tissues removed from anorganism, e.g. biopsy material.

Among certain embodiment of the present invention is a method of using apolypeptide, protein, pharmaceutical composition, and/or diagnosticcomposition of the present invention to detect the presence of a celltype for the purpose of information gathering regarding diseases,conditions and/or disorders. The method comprises contacting a cell witha diagnostically sufficient amount of a cytotoxic molecule to detect thecytotoxic molecule by an assay or diagnostic technique. The phrase“diagnostically sufficient amount” refers to an amount that providesadequate detection and accurate measurement for information gatheringpurposes by the particular assay or diagnostic technique utilized.Generally, the diagnostically sufficient amount for whole organism invivo diagnostic use will be a non-cumulative dose of between 0.1 mg to100 mg of the detection promoting agent linked cell-targeted molecule ofthe invention per kg of subject per subject. Typically, the amount ofpolypeptide or cell-targeted molecule of the invention used in theseinformation gathering methods will be as low as possible provided thatit is still a diagnostically sufficient amount. For example, for in vivodetection in an organism, the amount of polypeptide, protein, orpharmaceutical composition of the invention administered to a subjectwill be as low as feasibly possible.

The cell-type specific targeting of polypeptides and cell-targetedmolecules of the present invention combined with detection promotingagents provides a way to detect and image cells physically coupled withan extracellular target biomolecule of a binding region of the moleculeof the invention. Imaging of cells using the polypeptides orcell-targeted molecules of the present invention may be performed invitro or in vivo by any suitable technique known in the art. Diagnosticinformation may be collected using various methods known in the art,including whole body imaging of an organism or using ex vivo samplestaken from an organism. The term “sample” used herein refers to anynumber of things, but not limited to, fluids such as blood, urine,serum, lymph, saliva, anal secretions, vaginal secretions, and semen,and tissues obtained by biopsy procedures. For example, variousdetection promoting agents may be utilized for non-invasive in vivotumor imaging by techniques such as magnetic resonance imaging (MRI),optical methods (such as direct, fluorescent, and bioluminescentimaging), positron emission tomography (PET), single-photon emissioncomputed tomography (SPECT), ultrasound, x-ray computed tomography, andcombinations of the aforementioned (see, Kaur S et al., Cancer Lett 315:97-111 (2012), for review).

Among certain embodiment of the present invention is a method of using apolypeptide, protein, or pharmaceutical composition of the presentinvention as a diagnostic composition to label or detect the interiorsof cancer, tumor, and/or immune cell types (see e.g., Koyama Y et al.,Clin Cancer Res 13: 2936-45 (2007); Ogawa M et al., Cancer Res 69:1268-72 (2009); Yang L et al., Small 5: 235-43 (2009)). Based on theability of certain polypeptides, proteins, and pharmaceuticalcompositions of the invention to enter specific cell types and routewithin cells via retrograde intracellular transport, the interiorcompartments of specific cell types are labeled for detection. This canbe performed on cells in situ within a patient or on cells and tissuesremoved from an organism, e.g. biopsy material.

Diagnostic compositions of the present invention may be used tocharacterize a disease, disorder, or condition as potentially treatableby a related pharmaceutical composition of the present invention.Certain compositions of matter of the present invention may be used todetermine whether a patient belongs to a group that responds to atherapeutic strategy which makes use of a compound, composition orrelated method of the present invention as described herein or is wellsuited for using a delivery device of the invention.

Diagnostic compositions of the present invention may be used after adisease, e.g. a cancer, is detected in order to better characterize it,such as to monitor distant metastases, heterogeneity, and stage ofcancer progression. The phenotypic assessment of disease disorder orinfection can help prognostic and prediction during therapeutic decisionmaking. In disease reoccurrence, certain methods of the invention may beused to determine if local or systemic problem.

Diagnostic compositions of the present invention may be used to assessresponses to therapeutic(s) regardless of the type of therapeutic, e.g.small molecule drug, biological drug, or cell-based therapy. Forexample, certain embodiments of the diagnostics of the invention may beused to measure changes in tumor size, changes in antigen positive cellpopulations including number and distribution, or monitoring a differentmarker than the antigen targeted by a therapy already being administeredto a patient (see Smith-Jones P et al., Nat. Biotechnol 22: 701-6(2004); Evans M et al., Proc. Natl. Acad. Sci. U.S.A. 108: 9578-82(2011)).

Certain embodiments of the method used to detect the presence of a celltype may be used to gather information regarding diseases, disorders,and conditions, such as, for example bone cancer (such as multiplemyeloma or Ewing's sarcoma), breast cancer, central/peripheral nervoussystem cancer (such as brain cancer, neurofibromatosis, orglioblastoma), gastrointestinal cancer (such as stomach cancer orcolorectal cancer), germ cell cancer (such as ovarian cancers andtesticular cancers, glandular cancer (such as pancreatic cancer,parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroidcancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer,or pharyngeal cancer), hematological cancers (such as leukemia,lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancerand bladder cancer), liver cancer, lung/pleura cancer (such asmesothelioma, small cell lung carcinoma, or non-small cell lungcarcinoma), prostate cancer, sarcoma (such as angiosarcoma,fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma), skin cancer (suchas basal cell carcinoma, squamous cell carcinoma, or melanoma), uterinecancer, AIDS, amyloidosis, ankylosing spondylitis, asthma, autism,cardiogenesis, Crohn's disease, diabetes, erythematosus, gastritis,graft rejection, graft-versus-host disease, Grave's disease, Hashimoto'sthyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupuserythematosus, lymphoproliferative disorders, multiple sclerosis,myasthenia gravis, neuroinflammation, polyarteritis, psoriasis,psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock,Sjorgren's syndrome, systemic lupus erythematosus, ulcerative colitis,vasculitis, cell proliferation, inflammation, leukocyte activation,leukocyte adhesion, leukocyte chemotaxis, leukocyte maturation,leukocyte migration, neuronal differentiation, acute lymphoblasticleukemia (ALL), T acute lymphocytic leukemia/lymphoma (ALL), acutemyelogenous leukemia, acute myeloid leukemia (AML), B-cell chroniclymphocytic leukemia (B-CLL), B-cell prolymphocytic lymphoma, Burkitt'slymphoma (BL), chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML-BP), chronic myeloid leukemia (CML), diffuse large B-celllymphoma, follicular lymphoma, hairy cell leukemia (HCL), Hodgkin'sLymphoma (HL), intravascular large B-cell lymphoma, lymphomatoidgranulomatosis, lymphoplasmacytic lymphoma, MALT lymphoma, mantle celllymphoma, multiple myeloma (MM), natural killer cell leukemia, nodalmarginal B-cell lymphoma, Non-Hodgkin's lymphoma (NHL), plasma cellleukemia, plasmacytoma, primary effusion lymphoma, pro-lymphocyticleukemia, promyelocytic leukemia, small lymphocytic lymphoma, splenicmarginal zone lymphoma, T-cell lymphoma (TCL), heavy chain disease,monoclonal gammopathy, monoclonal immunoglobulin deposition disease,myelodusplastic syndromes (MDS), smoldering multiple myeloma, andWaldenstrom macroglobulinemia.

In certain embodiments, the polypeptides and cell-targeted molecules ofthe present invention, or pharmaceutical compositions thereof, are usedfor both diagnosis and treatment, or for diagnosis alone. In somesituations, it would be desirable to determine or verify the HLAvariant(s) and/or HLA alleles expressed in the subject and/or diseasedtissue from the subject, such as, e.g., a patient in need of treatment,before selecting a polypeptide or cell-targeted molecule of theinvention for treatment.

The present invention is further illustrated by the followingnon-limiting examples of 1) CD8+ T-cell hyper-immunized and/orB-cell/CD4+ T-cell de-immunized polypeptides, 2) CD8+ T-cell epitopepresenting toxin-derived polypeptides, and 3) selectively cytotoxic,cell-targeted proteins comprising the aforementioned polypeptides andcapable of specifically targeting certain cell types.

EXAMPLES

The following examples demonstrate certain embodiments of the presentinvention. However, it is to be understood that these examples are forillustration purposes only and do not intend, nor should any beconstrued, to be wholly definitive as to conditions and scope of thisinvention. The experiments in the following examples were carried outusing standard techniques, which are well known and routine to those ofskill in the art, except where otherwise described in detail.

The presentation of a T-cell immunogenic epitope peptide by the MHCclass I system targets the presenting cell for killing by CTL-mediatedlysis and also triggers immune stimulation in the localmicroenvironment. By engineering immunogenic epitope sequences withintoxin effector polypeptide components of target-cell-internalizingtherapeutics, the targeted delivery and presentation ofimmuno-stimulatory antigens may be accomplished. The presentation ofimmuno-stimulatory non-self antigens, such as e.g. known viral antigenswith high immunogenicity, by target cells signals to other immune cellsto destroy the target cells as well as to recruit more immune cells tothe area.

In the examples, T-cell epitopes were embedded or inserted into Shigatoxin effector polypeptides and diphtheria toxin effector polypeptides,which may serve as components of target-cell-internalizing molecules, byengineering internal regions to comprise one or more T-cell epitopes.Thus, there is no terminal fusion of an additional amino acid residue,peptide, or polypeptide component to the starting polypeptide.

In the examples, most of the T-cell epitopes were embedded into toxineffector polypeptide components of target-cell-internalizing moleculesby engineering multiple amino acid substitutions but without changingthe total number of amino acid residues in the exemplary toxin effectorpolypeptides as compared to the parental toxin effector polypeptide.Thus, for all of the diphtheria toxin effector polypeptides and most ofthe Shiga toxin effector polypeptides tested in the Examples, there wasno insertion of additional amino acids but rather only substitutions forexisting amino acids resulting in the maintenance of the original lengthof the parental polypeptide.

Novel toxin-derived effector polypeptides, which can function ascomponents of cell-targeted molecules (such as e.g. immunotoxins andligand-toxin fusions), were created which can promote cellularinternalization, sub-cellular routing to the cytosol, and delivery ofthe T-cell epitope to the cytosol for presentation by the MHC I classpathway to the target cell surface to signal to CTLs.

Certain novel toxin-derived effector polypeptides were also de-immunizedby embedding or inserting a T-cell epitope in a B-cell epitope regionusing one or more methods of the present invention. In order tosimultaneously de-immunize and provide for T-cell epitope presentationon the target cell surface within the same toxin polypeptide region,predicted B-cell epitope regions were disrupted by replacing them withknown T-cell epitopes predicted to bind to MHC Class I molecules. Aminoacid sequences from toxin-derived polypeptides were analyzed to predictantigenic and/or immunogenic B-cell epitopes in silico. Various T-cellepitope embedded, toxin-derived polypeptides were experimentally testedfor retention of toxin effector functions.

The preservation of toxin effector functions of exemplary T-cell epitopepresenting toxin effector polypeptides of the invention were tested andcompared to toxin effector polypeptides comprising wild-type toxinpolypeptide sequences, referred to herein as “wild-type” or “WT,” whichdid not comprise any internal modification or mutation to the toxineffector region.

The following examples of exemplary CD8+ T-cell epitope presenting Shigatoxin-derived polypeptides of the invention demonstrate methods ofsimultaneously providing for T-cell epitope delivery for MHC class Ipresentation while retaining one or more Shiga toxin effector functions.Further, the following examples of exemplary CD8+ T-cell epitopepresenting and/or B-cell/CD4+ T-cell de-immunized Shiga toxin-derivedpolypeptides of the invention demonstrate methods of simultaneouslyproviding for 1) T-cell epitope delivery for MHC class I presentation,2) retaining one or more toxin effector functions, and 3)de-immunization of the toxin effector region.

The exemplary cell-targeted molecules of the invention bound to targetbiomolecules expressed by targeted cell types and entered the targetedcells. The internalized exemplary cell-targeted proteins of theinvention effectively routed their de-immunized toxin effector regionsto the cytosol and effectively delivered immunogenic T-cell epitopes tothe target cells' MHC class I pathway resulting in presentation of theT-cell epitope peptide on the surface of target cells regions.

Example 1 Embedding or Inserting T-Cell Epitopes within PolypeptideComponents of Cell-Targeting Molecules

In this example, T-cell epitope sequences were selected from human viralproteins and embedded or inserted into Shiga toxin effectorpolypeptides. In some variants, the T-cell epitope was embedded orinserted into B-cell epitope regions in order to disrupt nativelyoccurring B-cell epitopes. In other variants, the T-cell epitope isembedded into regions not predicted to contain any B-cell epitopes and,thus, these modifications are not predicted to disrupt any dominantB-cell epitopes. In some of the above variants, the T-cell epitope isembedded into regions predicted to disrupt catalytic activity.

A. Selecting T-Cell Epitope Peptides for Embedding or Insertion

In this example, known T-cell epitope peptides were selected forembedding and inserting into Shiga toxin effector regions which have theintrinsic ability to intracellularly route to the cytosol. For example,there are many known immunogenic viral proteins and peptide componentsof viral proteins from human viruses, such as human influenza A virusesand human CMV viruses. Immunogenic viral peptides were chosen that arecapable of binding to human MHC class I molecules and/or eliciting humanCTL-mediated responses.

Seven peptides predicted to be T-cell epitopes (SEQ ID NOs:4-10) werescored for the ability to bind to common human MHC class I humanleukocyte antigen (HLA) variants encoded by the more prevalent allelesin human populations using the Immune Epitope Database (IEDB) AnalysisResource MHC-I binding prediction's consensus tool and recommendedparameters (Kim Y et al., Nucleic Acids Res 40: W252-30 (2012)). TheIEDB MHC-I binding prediction analysis predicted the “ANN affinity”—anestimated binding affinity between the input peptide and the selectedhuman HLA variant where IC₅₀ values less than 50 nanomolar (nM) areconsidered high affinity, IC₅₀ values between 50 and 500 nM areconsidered intermediate affinity, and IC₅₀ values between 500 and 5000nM are considered low affinity. The IEDB MHC-I binding predictionanalysis indicated the best binders by the lowest percentile ranks.Table 1 shows the IEDB MHC-I binding prediction percentile rank andpredicted binding affinity of the seven tested T cell epitope-peptides(SEQ ID NOs:4-10) binding to the selected human HLA variants.

TABLE 1 Predictions for Various Viral Protein-Derived T-Cell EpitopesBinding to Various Human MHC Class I Complexes IEDB MHC-I bindingprediction T-cell epitope Percentile Predicted Name Sequence HLA AlleleRank Affinity F2 GILGFVFTL HLA-A*32:01 0.80 intermediate HLA-A*02:010.80 high HLA-A*02:06 2.20 high F2-2 DILGFVFTL HLA-A*32:01 1.40intermediate HLA-A*02:01 4.60 low HLA-A*02:06 9.55 intermediate F2-3DILGFDFTL HLA-A*32:01 2.80 low HLA-A*02:01 8.20 low HLA-A*02:06 11.25low F2-4 GILGDVFTL HLA-A*02:01 1.40 high HLA-A*02:06 2.40 highHLA-A*32:01 3.10 low F3 ILRGSVAHK HLA-A*03:01 0.25 high HLA-A*30:01 0.70high HLA-A*31:01 4.25 intermediate F3-4 ILRFSVAHK HLA-A*03:01 0.25 highHLA-A*30:01 0.80 high HLA-A*31:01 3.30 intermediate C2 NLVPMVATVHLA-A*02:03 0.30 high HLA-A*02:01 1.00 high HLA-A*02:06 1.10 high

The results of the IEDB MHC-I binding prediction analysis show that somepeptides were predicted to bindwith high affinity to multiple human MHCclass I molecules, whereas other peptides were predicted to bind withmore moderate affinities to the analyzed human MHC class I molecules.

B. Identifying B-Cell Epitope Regions in Toxins and Toxin EffectorPolypeptides

Toxin derived polypeptides with intrinsic subcellular routingcharacteristics suitable for proteasome delivery were chosen forde-immunization in order to reduce the possibility of undesirable immuneresponses after administration to chordate, such as, e.g., theproduction of anti-toxin antibodies. Amino acid sequences from toxinsand toxin-derived polypeptides were analyzed to predict antigenic and/orimmunogenic B-cell epitopes in silico.

Polypeptide effectors derived from both a Shiga toxin and a diphtheriatoxin were analyzed for B-cell epitopes.

Shiga Toxin Derived Effector Polypeptides

First, B-cell epitope regions were identified within Shiga toxin ASubunits. Computational methods were utilized to predict antigenicand/or immunogenic B-cell epitopes in Shiga toxin A subunit sequenceswith the potential to elicit responses by mammalian immune systems afteradministration.

Linear B-cell epitopes were predicted within the A Subunits of Shigatoxins using the polypeptide sequence and 3D structural data ofShiga-Like Toxin Chain A (PDB ID: 1DM0_A) and the REVEAL® systemprovided by ProImmune, Inc. (Sarasota, Fla., U.S.). In parallel, B-cellepitopes were predicted within the amino acid sequences of the A Subunitof Shiga toxins using the BcePred webserver (Saha S, Raghava G, LectureNotes in Comput Sci 3239: 197-204 (2004)), Bepipred Linear EpitopePrediction (Larsen J et al., Immunome Res 2: 2 (2006)), ElliPro Antibodyepitope prediction (Haste Andersen P et al., Protein Sci 15: 2558-67(2006); Ponomarenko J, Bourne P, BMC Struct Biol 7: 64 (2007)), and/orthe Epitopia server (Rubinstein N et al., BMC Bioinformatics 10: 287(2009)). The Epitopia server prediction was used to identify immunogenicB-cell epitopes as any stretch of linear amino acid residues comprisinga majority of residues predicted on Epitopia's immunogenicity scale tobe “high” (scored as 4 or 5). The various computational methods revealedsimilar predictions for B-cell epitope regions in the three prototypicalShiga toxin A Subunits (Tables 2-4).

TABLE 2 B-Cell Epitope Predictions for the Mature, Native A Subunit ofShiga-like Toxin 1 (SEQ ID NO: 1) Epitope natively positioned amino acidpositions Region REVEAL BcePred Bepipred ElliPro Epitopia 1  1-15 229-35 28-34 27-37 18-23 3 42-48 39-46 43-47 44-49 4 58-66 55-61 56-6457-66 52-62 5  96-103 105-111 100-115  96-110 94-102, 109- 114 6 144-151141-147 147-151 144- 153 7 183-189 181-187 183-185 180- 179-188 190 8211-219 211-220 9 243-251 243- 245-259 257 10 257-268 261-267 254-268 11289-293 285-291 262- 262-281 293

TABLE 3 B-Cell Epitope Predictions for the Mature, Native A Subunit ofShiga Toxin (SEQ ID NO: 2) natively positioned amino acid positionsREVEAL BcePred Bepipred ElliPro 29-35 28-34 27-37 42-48 39-46 44-4758-66 55-61 56-64 57-66  96-103 105-111 100-115  96-110 144-151 141-147147-151 144-153 183-189 181-187 183-185 180-190 211-219 243-251 243-257257-268 261-267 254-268 289-293 285-291 262-293

TABLE 4 B-Cell Epitope Predictions for the Mature, Native A Subunit ofShiga-like Toxin 2 (SEQ ID NO: 3) natively positioned amino acidpositions BcePred Bepipred ElliPro  3-11  8-14 29-35 28-36 26-37 42-4857-62 56-66 108-115 109-115  96-110 141-156 140-153 179-188 180-191210-218 210-217 240-257 244-258 241-255 262-278 281-297

In addition to Shiga toxin-derived toxin effector polypeptides, whichare capable of inducing cellular internalization and directing their ownsubcellular routing to the cytosol, cytosolic routing effector regionsfrom other proteins may be chosen as a source for polypeptides to modifyinto a polypeptide of the present invention, such as, e.g., from otherABx and/or RIP toxins.

Diphtheria Toxin Derived Effector Polypeptides

Diphtheria toxins have been used to design immunotoxins and ligand-toxinfusion molecules wherein the diphtheria derived component can providecellular internalization and cytosolic routing effector functions. Acomputational method was utilized to predict antigenic and/orimmunogenic B-cell epitope regions in the diphtheria toxin A subunitwith the potential to elicit responses in mammalian immune systems.B-cell epitope regions were predicted in the A Subunit of diphtheriatoxin (SEQ ID NO:44) using the BcePred webserver (Saha S, Raghava G,Lecture Notes in Comput Sci 3239: 197-204 (2004)). This computationalmethod revealed seven putative B-cell epitope regions in theprototypical Diphtheria Toxin A Subunit (Table 5). In addition, theImmune Epitope Database (IEDB) curated by the National Institutes ofAllergy and Infectious Diseases of the U.S. (NIAID) is said to provideall experimentally characterized B-cell nd T-cell epitopes of diphtheriatoxins. Currently, the IEDB provides 7 epitopes with at least onepositive measurement regarding peptidic epitopes related to thediphtheria toxin A subunit and the diphtheria toxin effector polypeptideSEQ ID NO:44 used in the Examples (see Table 5 and region 182-201 and225-238 of SEQ ID NO:44).

TABLE 5 B-Cell Epitope Predictions for the Mature, Native A Subunit ofDiphtheria Toxin (SEQ ID NO: 44) Natively positioned amino acids EpitopeRegion BcePred IEDB 1  3-10 2 15-31 3 33-43 32-54 4 71-77 5  93-113 6125-131 7 138-146 141-167 8 165-175 141-167 9 185-191 181-193

C. Identifying CD4+ T-Cell Epitope Regions in Toxins and Toxin EffectorPolypeptides

The Shiga toxin A subunit was analyzed for the presence of any CD4+T-cell epitopes. T-cell epitopes were predicted for the mature A Subunitof Shiga-like toxin 1 (SEQ ID NO:1) by the REVEAL™ Immunogenicity System(IS) T-cell assay performed by ProImmune Inc. (Sarasota, Fla., U.S.).This assay uses multiple overlapping peptide sequences from the subjectprotein to test for the elicitation of any immune response by CD4+T-cells from healthy donor cell samples depleted of CD8+ T-cells. Therewere seven T-cell epitope regions identified using this assay at thefollowing natively positioned groups of amino acid residues: CD4+ T-cellepitope region #1: 4-33, CD4+ T-cell epitope region #2: 34-78, CD4+T-cell epitope region #3: 77-103, CD4+ T-cell epitope region #4:128-168, CD4+ T-cell epitope region #5: 160-183, CD4+ T-cell epitoperegion #6: 236-258, and CD4+ T-cell epitope region #7: 274-293.

The diphtheria toxin A subunit and a wild-type (WT), diphtheria toxineffector polypeptide used as a parental polypeptide for generation ofthe diphtheria toxin effector polypeptides in the Examples, wereinvestigated on NIAD's IEDB for T-cell epitopes. Currently, the IEDBprovides over 25 peptidic epitopes with at least one positivemeasurement regarding T-cell immunogenic related to the diphtheria toxinA subunit and the diphtheria toxin effector polypeptides in theExamples. There were several T-cell epitope regions identified by theIEDB in diphtheria toxins, such as, e.g., the following regionscorresponding to overlapping immunogenic peptides in the polypeptide ofSEQ ID NO:45 at amino acid residue positions: 2-21, 22-41, 32-71, 72-91,82-221, 212-231, 232-251, and 251-301.

D. Generating Toxin Effector Polypeptides with Embedded or InsertedT-Cell Epitopes Disrupting Endogenous B-Cell Epitope Regions and/orEndogenous CD4+ T-Cell Epitope Regions

Exemplary toxin-derived effector polypeptides of the invention werecreated using both a Shiga toxin and a diphtheria toxin.

Shiga Toxin Derived Effector Polypeptides

A nucleic acid encoding a cytotoxic protein comprising a Shiga toxineffector region and an immunoglobulin-type binding region for celltargeting was produced using techniques known in the art. The Shigatoxin effector region in the parental cytotoxic protein of this examplecomprised amino acids 1-251 of SEQ ID NO:1.

Using standard techniques known in the art, a series of mutations wereengineered into the nucleic acid encoding the parental cytotoxic proteinand variants of the cytotoxic protein were produced which comprisedmultiple amino acid substitutions as compared to the parental cytotoxicprotein. The mutations were selected to disrupt at least one predictedB-cell epitope region described in Table 2 by embedding at least oneT-cell epitope peptide described in Table 1. For most of the exemplarypolypeptides of the invention described in the Examples, the amino acidsequence for each T-cell epitope was embedded by manipulating thenucleic acid sequences encoding the region of interest such that thetotal number of encoded amino acid residues in the variants remainedunchanged from the total number of amino acid residues in the parentalcytotoxic protein. Ten different polynucleotides were generated whicheach encoded a different exemplary cytotoxic, cell-targeted protein ofthe invention comprising a different exemplary Shiga toxin effectorpolypeptide component of the invention. These exemplary polynucleotideswere used to produce ten exemplary cytotoxic, cell-targeted proteins ofthe invention using standard techniques known in the art. In certainexperiments, the full-length coding sequence of the cytotoxic protein ofthis example began or ended with a polynucleotide encoding a Strep-tag®II to facilitate detection and purification. Proteins were purifiedusing methods known to the skilled worker.

Eleven cytotoxic proteins were derived from the parental cytotoxicprotein, each comprising an exemplary Shiga toxin effector polypeptideof the invention (selected from SEQ ID NOs: 11-21) and a disruption ofat least one of the predicted B-cell epitope regions in Table 2 usingone of the T-cell epitopes described in Table 1. The exact modificationto the parental Shiga toxin effector polypeptide for each of the elevencytotoxic proteins is shown in Table 6. Table 6 lists the sequence ofeach embedded T-cell epitope, the native position in the Shiga toxin ASubunit of the modification, and the disrupted stretch of amino acids inthe B-cell epitope region.

TABLE 6 Exemplary Shiga Toxin Effector Poly peptides with T-CellEpitopes Embedded or Inserted into B-Cell Epitope Regions and/or CD4+T-Cell Epitope Regions Position T-Cell B-Cell (native residue EpitopeT-Cell Epitope Epitope B-Cell Epitope positions) Name Embedded RegionRegion Replaced  4-12 F3-4 ILRFSVAHK 1 TLDFSTAKT 43-51 C2 NLVPMVATV 3SGSGDNLFA 44-52 F2 GILGFVFTL 3 GSGDNLFAV 44-52 C2 NLVPMVATV 3 GSGDNLFAV53-61 F2-2 DILGFVFTL 4 DVRGIDPEE 53-61 F2-3 DILGFDFTL 4 DVRGIDPEE 53-61C2 NLVPMVATV 4 DVRGIDPEE 104-112 C2 NLVPMVATV 5 TAVTLSGDS 180-188 F2-4GILGDVFTL 7 TTLDDLSGR 53-61 F2 GILGFVFTL 4 DVRGIDPE 245/246 F3 ILRGSVAHK9 none (inserted at 246)

The first nine cytotoxic proteins each comprised a Shiga toxin effectorpolypeptide comprising an embedded T-cell epitope (see Table 6)—meaningwithout any change to the overall total number of amino acid residues inthe Shiga toxin effector polypeptide component of the parental cytotoxicprotein. Each of the first nine modifications listed in Table 6exemplifies an embedded T-cell epitope which disrupts a B-cell epitoperegion. As these nine modifications are exact replacements, the T-cellepitope sequence and B-cell epitope region sequence disrupted areidentical in length and match one-for-one each amino acid as listed inorder from amino-terminus to carboxy-terminus. The tenth Shiga toxineffector polypeptide in Table 6, 53-61-F2, comprises both a partialreplacement and an insertion of one amino acid at position 61 whichshifts the remaining carboxy-terminal, wild-type, amino acid residues byone position. The eleventh Shiga toxin effector polypeptide in Table 6is a complete insertion of the entire T-cell epitope between nativelypositioned amino acid residues 245 and 246. This insertion lies withinB-cell epitope region #9 natively positioned at amino acids 243-259 ofSLT-1A.

Computational analysis in silico predicted that at least one B-cellepitope present in the wild-type Shiga toxin was eliminated for eight ofthe T-cell epitope embedded or inserted Shiga toxin effector polypeptidevariants, and no new B-cell epitopes were predicted to be generated byembedding or inserting a T-cell epitope in any of the exemplary Shigatoxin effector polypeptides in Table 6 (see also Example 3, infra).

In addition, the Shiga toxin effector polypeptides represented by SEQ IDNOs:11-17 and 19-21 each comprise a disruption of a predicted endogenousCD4+ T-cell epitope(s).

Diphtheria Toxin Derived Effector Polypeptides

Similar to the above descriptions of modifying Shiga toxin-derivedpolypeptides, T-cell epitopes were embedded into diphtheriatoxin-derived polypeptides with proteasome delivery effector function tocreate exemplary T-cell epitope embedded, diphtheria toxin effectorpolypeptides of the invention. The T-cell epitopes were selected from apeptide in Table 1 and embedded to disrupt at least one predicted B-cellepitope region described in Table 5.

All the diphtheria toxin-derived polypeptides of this example comprisedthe catalytic domain from the diphtheria toxin A Subunit continuous withthe translocation domain from the diphtheria toxin B Subunit, a furincleavage motif between the A and B subunit derived, toxin effectorpolypeptide regions, and a predicted disulfide bond between cysteines inthe A and B subunit derived, toxin effector polypeptide regions. Thus,the diphtheria toxin-derived polypeptides in this example comprise botha proteasome delivery effector region and ribotoxic toxin effectorpolypeptide. The polypeptide sequences of exemplary, T-cell epitopeembedded, diphtheria toxin effector polypeptides of the invention areprovided as SEQ ID NOs: 46, 47, and 48.

Using standard techniques, a series of mutations were made in thediphtheria toxin effector polypeptide in order to embed a T-cell epitopein a position overlapping a predicted B-cell epitope region (see Table5). Table 7 shows examples of T-cell epitope embedded, diphtheria toxineffector polypeptides by denoting the position of embedded T-cellepitope based on the native diphtheria toxin polypeptide sequence in SEQID NO:44, the T-cell epitope name, the T-cell epitope peptide sequence,the predicted B-cell epitope region disrupted, and the replaced aminoacid sequence in the native diphtheria toxin polypeptide sequence.

TABLE 7 Exemplary Diphtheria Toxin Effector Polypeptides with T-CellEpitopes Embedded into B-Cell Epitope Regions B-Cell Epitope PositionB-Cell Prediction (native T-Cell T-Cell Epitope Epitope B-Cell Epitopeoriginal neo- positions) Epitope Embedded Region Region Replaced epitopeepitope 34-42 F2 GILGFVFTL 2 GIQKPKSGT eliminated none 69-77 C2NLVPMVATV 3 NENPLSGKA eliminated none 168-176 F3 ILRGSVAHK 6 ETRGKRGQDeliminated none

The T-cell epitope embedded, diphtheria toxin effector polypeptidevariants (SEQ ID NOs: 46, 47, and 48) were analyzed for any change inthe predicted B cell epitopes as described above. In all three T-cellepitope embedded, diphtheria toxin effector polypeptide variants, thepredicted B-cell epitope in the wild-type diphtheria toxin amino acidsequence was eliminated, and no new B-cell epitopes were predicted(Table 7).

Three T-cell epitope embedded, diphtheria toxin effector polypeptidevariants (SEQ ID NOs: 46, 47, and 48), and the parental diphtheria toxineffector polypeptide comprising only wild-type toxin amino acidsequences (SEQ ID NO:45), were each designed with an amino-terminalmethionine and a carboxy-terminal polyhistidine-tag (6×His tag) tofacilitate expression and purification. Both exemplary T-cell epitopeembedded, diphtheria toxin effector polypeptide variants of theinvention and the parental diphtheria toxin effector polypeptidecomprising only wild-type toxin amino acid sequences were produced by abacterial expression system known in the art and purified underconditions known in the art, such as, e.g., nickel-nitrilotriacetic acid(Ni-NTA) resin chromatography.

E. Generating Shiga Toxin Effector Polypeptides with Embedded T-CellEpitopes which do not Disrupt any B-Cell Epitope Region

Recognizing all the B-cell epitope region predictions from all themethods described in the Examples (Table 2), regions of SLT-1A that werenot predicted to contain any B-cell epitope were identified. T-cellepitope peptide sequences from Table 1 are embedded in those regionsidentified to lack B-cell epitopes by replacing the native amino acidsby substitutions to create three different exemplary Shiga toxineffector polypeptides of the invention as shown in Table 8. Table 8shows the identified regions in the mature, native SLT-1A polypeptidesequence and the replacement T-cell epitope sequences constructed intothe Shiga toxin effector polypeptides (see SEQ ID NOs: 22-39).

TABLE 8 T-Cell Epitopes Embedded Outside B-Cell Epitope Regions in Shiga Toxin Effector Polypeptides Position T-Cell T-Cell(native residue Epitope Epitope WT Region positions) Name Embeddedreplaced 66-74 F2 GILGFVFTL NLRLIVERN 75-83 F2 GILGFVFTL NLYVTGFVN157-165 F2 GILGFVFTL AMLRFVTVT 164-172 F2 GILGFVFTL VTAEALRFR 221-229 F2GILGFVFTL VGRISFGSI 231-239 F2 GILGFVFTL AILGSVALI 66-74 F3 ILRGSVAHKNLRLIVERN 75-83 F3 ILRGSVAHK NLYVTGFVN 157-165 F3 ILRGSVAHK AMLRFVTVT164-172 F3 ILRGSVAHK VTAEALRFR 221-229 F3 ILRGSVAHK VGRISFGSI 231-239 F3ILRGSVAHK AILGSVALI 66-74 C2 NLVPMVATV NLRLIVERN 75-83 C2 NLVPMVATVNLYVTGFVN 157-165 C2 NLVPMVATV AMLRFVTVT 164-172 C2 NLVPMVATV VTAEALRFR221-229 C2 NLVPMVATV VGRISFGSI 231-239 C2 NLVPMVATV AILGSVALI

The Shiga toxin effector polypeptide sequences comprising, as exactreplacements, the embedded T-cell epitopes in Table 8 were analyzedusing the BcePred program. None of the embedded T-cell epitope exactreplacements in Table 8 disrupted any of the six epitope regionspredicted by that program. One of the embedded T-cell epitopereplacement sequences in Table 8, variant 75-83-F3, resulted in theprediction of a new B-cell epitope. Embedding T-cell epitopes near theregions (66-74) and/or (157-165) may interfere with the Shiga toxineffector function of catalytic activity because of their proximity to atleast one amino acid known to be required for SLT-1A catalytic activity(e.g. Y77 and E167).

In addition, the Shiga toxin effector polypeptides represented by SEQ IDNOs: 22-39 all comprise a disruption of a predicted endogenous CD4+T-cell epitope(s) except for the polypeptides with heterologous T-cellepitopes embedded at position 221-229, which are represented by SEQ IDNOs: 26, 32, and 38.

F. Generating Toxin Effector Polypeptides with Embedded T-Cell Epitopeswhich Disrupt Toxin Catalytic Function

The most critical residues for enzymatic activity of the Shiga toxin ASubunits include tyrosine-77 (Y77) and glutamate-167 (E167) (Di, Toxicon57: 535-39 (2011)). T-cell epitope peptide sequences from Table 1 areembedded into Shiga toxin effector polypeptides such that either Y77 orE167 is mutated in order to reduce or eliminate Shiga toxin enzymaticactivity. Six different exemplary Shiga toxin effector polypeptides ofthe invention comprising a heterologous T-cell epitope disrupting acatalytic amino acid residue are shown in Table 9. Table 9 shows theposition of the embedded T-cell epitopes in the mature, native SLT-1Apolypeptide sequence, the replacement T-cell epitope sequences which areembedded, the replaced sequences in the mature, native SLT-1Apolypeptide sequence, and a resulting catalytic residue disruption (seealso SEQ ID NOs: 23, 29, 40, 41, 42, and 43).

TABLE 9 T-Cell Epitopes Embedded in Shiga ToxinEffector Polypeptides to Inactivate Shiga Toxin Catalytic FunctionPosition (native T-Cell T-Cell Catalytic residue Epitope EpitopeWT Region Residue positions) name Embedded Replaced Change 75-83 C2NLVPMVATV NLYVTGFVN Y77V 75-83 F3 ILRGSVAHK NLYVTGFVN Y77R 77-85 F2GILGFVFTL YVTGFVNRT Y77G 159-167 F2 GILGFVFTL LRFVTVTAE E167L 159-167 F3ILRGSVAHK LRFVTVTAE E167K 162-170 C2 NLVPMVATV VTVTAEALR E167V

All of the Shiga toxin effector polypeptides represented by SEQ ID NOs:23, 29, 40, 42, and 43 comprise disruptions of a predicted endogenousCD4+ T-cell epitope(s). In addition, among the exemplary Shiga toxineffector polypeptides with embedded T-cell epitopes which do not disruptany B-cell epitope region shown in Table 8, at least eight of themdisrupt a catalytic amino acid residue of the Shiga toxin effectorregion (see SEQ ID NOs: 23, 25, 29, 31 35, and 37).

In addition to embedding and inserting at a single site, multipleimmunogenic T-cell epitopes for MHC class I presentation are embeddedand/or inserted within the same Shiga toxin-derived polypeptides ordiphtheria toxin-derived polypeptides for use in the targeted deliveryof a plurality of T-cell epitopes simultaneously, such as, e.g.,disrupting a B-cell epitope region with a first embedded T-cell epitopeand disrupting a toxin catalytic function with a second embedded T-cellepitope. However, it should be noted that a single embedded T-cellepitope can simultaneously disrupt both a B-cell epitope region and atoxin catalytic function.

Example 2 Testing Toxin-Derived Effector Polypeptides for Retention ofRibotoxic Toxin Effector Function

Exemplary toxin-derived effector polypeptides of the invention weretested for retention of ribotoxic toxin effector function.

Shiga Toxin Derived Effector Polypeptides' Retention of Ribotoxicity

The retention of the enzymatic activity of the parental Shiga toxineffector polypeptide after embedding or inserting one or more T-cellepitopes was determined using a ribosome inhibition assay. The resultsof this assay in this example were based on performing the assay witheach Shiga toxin effector polypeptide as a component of a cytotoxicprotein. The specific cytotoxicities of different cytotoxic proteinscomprising different Shiga toxin effector polypeptides were measuredusing a tissue culture cell-based toxicity assay. The enzymatic andcytotoxic activities of the exemplary cytotoxic, cell-targeted proteinsof the invention were compared to the parental Shiga toxin effectorpolypeptide alone (no cell-targeting binding region) and a “WT”cytotoxic protein comprising the same cell-targeting domain (e.g.binding region comprising an immunoglobulin-type binding region capableof binding an extracellular target biomolecule with high affinity) butwith a wild-type Shiga toxin effector region (WT).

The ribosome inactivation capabilities of cytotoxic proteins comprisingembedded or inserted T-cell epitopes were determined using a cell-free,in vitro protein translation assay using the TNT® Quick CoupledTranscription/Translation kit (L1170 Promega Madison, Wis., U.S.). Thekit includes Luciferase T7 Control DNA (L4821 Promega Madison, Wis.,U.S.) and TNT® Quick Master Mix. The ribosome activity reaction wasprepared according to manufacturer's instructions. A series of 10-folddilutions of the protein to be tested, comprising either a mutated Shigatoxin effector polypeptide region or WT region, was prepared in anappropriate buffer and a series of identical TNT reaction mixturecomponents were created for each dilution. Each sample in the dilutionseries was combined with each of the TNT reaction mixtures along withthe Luciferase T7 Control DNA. The test samples were incubated for 1.5hours at 30 degrees Celsius (° C.). After the incubation, LuciferaseAssay Reagent (E1483 Promega, Madison, Wis., U.S.) was added to all testsamples and the amount of luciferase protein translation was measured byluminescence according to manufacturer's instructions. The level oftranslational inhibition was determined by non-linear regressionanalysis of log-transformed concentrations of total protein versusrelative luminescence units. Using statistical software (GraphPad Prism,San Diego, Calif., U.S.), the half maximal inhibitory concentration(IC₅₀) value was calculated for each sample using the Prism softwarefunction of log(inhibitor) vs. response (three parameters)[Y=Bottom+((Top−Bottom)/(1+10̂(X−Log IC₅₀)))] under the headingdose-response-inhibition. The IC₅₀ values were calculated for eachde-immunized protein comprising a B cell epitope replacement/disruptionShiga toxin effector polypeptide region and a control protein comprisinga wild-type Shiga toxin effector region (WT).

The exemplary Shiga toxin effector polypeptide regions of the inventionexhibited ribosome inhibition comparable to a wild-type Shiga toxineffector polypeptide (WT) as indicated in Table 10. As reported in Table10, any construct comprising a Shiga toxin effector polypeptide of theinvention which exhibited an IC₅₀ within 10-fold of the positive controlconstruct comprising a wild-type Shiga toxin effector region wasconsidered to exhibit ribosome inhibition activity comparable towild-type.

TABLE 10 Retention of Shiga Toxin Function(s): In vitro catalyticactivity and in vivo specific cytotoxicity of exemplary Shiga toxineffector polypeptides Exemplary Shiga Toxin Shiga Toxin FunctionsEffector Polypeptide Ribosome Position-T-Cell-Epitope InactivationSpecific Cytotoxicity 4-12-F3-4 comparable to WT comparable to WT43-51-C2 comparable to WT comparable to WT 44-52-F2 comparable to WTcomparable to WT 53-61-F2 comparable to WT comparable to WT 53-61-F2-2comparable to WT comparable to WT 53-61-F2-3 comparable to WT comparableto WT 53-61-C2 comparable to WT comparable to WT 104-112-C2 comparableto WT comparable to WT 180-188-F2-4 comparable to WT comparable to WT245-F3 comparable to WT comparable to WT

The retention of cytotoxicity by exemplary Shiga toxin effectorpolypeptides of the invention after T-cell epitope embedding/insertionwas determined by a cell-kill assay in the context of the Shiga toxineffector polypeptide as a component of a cytotoxic protein. Thecytotoxicity levels of proteins comprising Shiga toxin effectorpolypeptides, comprising an embedded or inserted T-cell epitope, weredetermined using extracellular target expressing cells as compared tocells that do not express a target biomolecule of the cytotoxicprotein's binding region. Cells were plated (2×10³ cells per well foradherent cells, plated the day prior to protein addition or 7.5×10³cells per well for suspension cells, plated the same day as proteinaddition) in 20 μL cell culture medium in 384-well plates. A series of10-fold dilutions of each protein comprising a mutated Shiga toxineffector polypeptide region to be tested was prepared in an appropriatebuffer, and then 5 μL of the dilutions or buffer control were added tothe cells. Control wells containing only media were used for baselinecorrection. The cell samples were incubated with the proteins or justbuffer for 3 days at 37° C. and in an atmosphere of 5% carbon dioxide(CO₂). The total cell survival or percent viability was determined usinga luminescent readout using the CellTiter-Glo® Luminescent CellViability Assay (G7573 Promega Madison, Wis., U.S.) according to themanufacturer's instructions.

The Percent Viability of experimental wells was calculated using thefollowing equation: (Test RLU−Average Media RLU)/(Average CellsRLU−Average Media RLU)*100. Log polypeptide concentration versus PercentViability was plotted in Prism (GraphPad Prism, San Diego, Calif., U.S.)and log (inhibitor) versus response (3 parameter) analysis was used todetermine the half-maximal cytotoxic concentration (CD₅₀) value for thetested proteins. The CD₅₀ was calculated for each protein comprising anexemplary Shiga toxin effector polypeptide of the invention in Table 10,positive-control cytotoxic protein comprising a wild-type Shiga toxineffector region, and the wild-type SLT-1 A Subunit alone (no targetingdomain)—both were considered WT positive controls.

The protein comprising exemplary Shiga toxin effector polypeptides ofthe invention exhibited cell-specific cytotoxicities comparable to awild-type (WT) Shiga toxin effector polypeptide as indicated in Table10. As reported in Table 10 with regard to specific cytotoxicity,“comparable to WT” means a protein comprising a Shiga toxin effectorpolypeptide, comprising an embedded or inserted T-cell epitope,exhibited a CD₅₀ to a target positive cell population within 10-fold ofa protein comprising a wild-type (WT) Shiga toxin effector region and/orless than 50-fold of the SLT-1A subunit alone.

In addition, the same protein constructs comprising exemplary Shigatoxin effector polypeptides of the invention exhibited specificcytotoxicity to biomolecular-target-expressing cells as compared tobiomolecular-target-negative cells (i.e. cells which did not express, ata cellular surface, the biomolecular target of the cell-target bindingregion of the protein construct). Thus, all the proteins comprising theexemplary Shiga toxin effector polypeptides in Table 10 were cytotoxicproteins exhibiting Shiga toxin effector functions comparable towild-type (WT), and each cytotoxic protein comprised a disruption in oneor more predicted, B-cell epitope regions.

Diphtheria Toxin Derived Effector Polypeptides' Retention ofRibotoxicity

The catalytic activity of exemplary, T-cell epitope embedded, diphtheriatoxin effector polypeptides were compared to diphtheria toxin effectorpolypeptides comprising only wild-type amino acid sequences, referred toherein as “wild-type” or “WT.” Both T-cell epitope embedded, diphtheriatoxin effector polypeptide variants retained ribosome inactivationactivity.

The retention of enzymatic activity of diphtheria toxin effectorpolypeptide variants with embedded T-cell epitopes in the context of acell-targeted molecule was tested using a ribosome inhibition assay anda wild-type (WT) diphtheria toxin effector polypeptide as a positivecontrol. The ribosome inactivation capabilities of these toxin effectorpolypeptides was determined using a cell-free, in vitro proteintranslation assay using the TNT® Quick Coupled Transcription/Translationkit (L1170 Promega Madison, Wis., U.S.) as described above unlessotherwise noted. First, the diphtheria toxin effector polypeptides werecleaved in vitro with furin (New England Biolabs, Ipswich, Mass., U.S.)under standard conditions. Then the cleaved proteins were diluted inbuffer to make a series of dilutions for each sample. Each dilution ineach series was combined with each of the TNT reaction mixtures alongwith the Luciferase T7 Control DNA and tested for ribosome inactivationactivity as described above.

The IC₅₀ was calculated, as described above, for the diphtheria toxineffector polypeptides. FIG. 2 and Table 11 show the results of this invitro assay for retention of diphtheria ribotoxic toxin effectorfunction by exemplary, T-cell embedded, diphtheria toxin effectorpolypeptides of the invention. The activity of the T-cell embedded,diphtheria toxin effector polypeptides was comparable to the wild-type(WT) positive control because the IC₅₀ values were within ten-fold ofthe wild-type diphtheria toxin effector polypeptide control (FIG. 2;Table 11).

TABLE 11 Retention of catalytic activity by exemplary T-cell epitopeembedded, Diphtheria toxin effector polypeptides Fold Change DiphtheriaToxin Effector Polypeptide IC₅₀ (μM) from WT Wild-type (WT) 1.80 1.0T-cell epitope embedded, diphtheria toxin 4.94 2.8 effector polypeptidevariant 34-42-F2 T-cell epitope embedded, diphtheria toxin 13.3 7.5effector polypeptide variant 168-176-F3 Exemplary Diphtheria ToxinEffector Diphtheria Toxin Function: Polypeptide Ribosome Inactivation34-42-F2 comparable to WT 168-176-F3 comparable to WT

Example 3 Testing the De-Immunization Effects of Disruption of B-CellEpitope Regions and CD4+ T-Cell Epitope Regions in T-Cell EpitopeEmbedded, Toxin Effector Polypeptides

The disruption of B-cell epitope regions in Shiga toxin effectorpolypeptides using embedded or inserted T-cell epitopes was tested forde-immunization by investigating levels of antigenicity and/orimmunogenicity compared to wild-type (WT) Shiga toxin effectorpolypeptides comprising only wild-type amino acid sequences.

Testing De-Immunization Via Western Analysis

To analyze de-immunization, the antigenicity or immunogenicity levels ofShiga toxin effector polypeptides was tested both in silico and byWestern blotting using pre-formed antibodies which recognize wild-typeShiga toxin effector polypeptides.

Each Shiga toxin effector polypeptide described in Table 6 (SEQ ID NOs:11-21) was checked for the disruption of predicted B-cell epitopes usingthe BcePred webserver using the following parameters: flexibilityreadout with the default settings of hydrophilicity 2, accessibility 2,exposed surface 2.4, antigenic propensity 1.8, flexibility 1.9, turns1.9, polarity 2.3, and combined 1.9 (Saha S, Raghava G, Lecture Notes inComput Sci 3239: 197-204 (2004)). Three predicted immunogenic epitoperegions identified in the wild-type SLT-1 A Subunit by other programs(see Table 2) were not predicted by the BcePred flexibility approachwith the default settings and, thus, could not be analyzed.

The T-cell epitope embedding or insertion in the following exemplaryShiga toxin effector polypeptides of the invention SEQ ID NOs: 11-21resulted in the elimination of the predicted B-cell epitope intended fordisruption without the introduction of any epitopes de novo(neo-epitopes) (Table 12). None of the tested exemplary Shiga toxineffector polypeptides of the invention resulted in the generation of anyde novo predicted B-cell epitopes using the BcePred flexibility approachwith the default settings (Table 12). Any B-cell epitope region notpredicted by the BcePred flexibility approach with the default settingswas indicated with “not identified” and the result after T-cell epitopeembedding or insertion was indicated with “N/A” to denote “notapplicable.

TABLE 12 Analysis of B-Cell Epitope Region Disruption byEmbedded or Inserted T-Cell Epitopes B-Cell Epitope Region DisruptionBcePred Flexibility B-Cell with T-Cell Epitope ReplacementEpitope Predictions T-Cell T-Cell B-Cell WT Shiga Modified EpitopeEpitope Epitope Region Toxin Sequence Shiga Toxin Neo-Epitope PositionEmbedded Disrupted (parental) Sequence Prediction  4-12 ILRFSVAHK 1 notN/A none identified 43-51 NLVPMVATV 3 39-46 eliminated none 44-52GILGFVFTL 3 39-46 eliminated none 44-52 NLVPMVATV 3 39-46 eliminatednone 53-61 GILGFVFTL 4 55-61 eliminated none 53-61 DILGFVFTL 4 55-61eliminated none 53-61 DILGFDFTL 4 55-61 eliminated none 53-61 NLVPMVATV4 55-61 eliminated none 104-112 NLVPMVATV 5 105-111 eliminated none180-188 GILGDVFTL 7 181-187 eliminated none 245/246 ILRFSVAHK 9 not N/Anone identified

The relative antigenicity levels of Shiga toxin effector polypeptideswas tested for de-immunization by Western blotting using pre-formedantibodies, both polyclonal and monoclonal antibodies, which recognizethe wild-type (WT) Shiga toxin effector polypeptides comprising aminoacids 1-251 of SEQ ID NO:1.

Western blotting was performed on cytotoxic proteins comprising a Shigatoxin effector polypeptide comprising either only a wild-type (WT) Shigatoxin sequence or one of various modified Shiga toxin sequencescomprising a B-cell epitope region disruption via replacement with aT-cell epitope (SEQ ID NO: 11-19). These cytotoxic proteins were loadedin equal amounts to replicate, 4-20% sodium dodecyl sulfate (SDS),polyacrylamide gels (Lonza, Basel, CH) and electrophoresed underdenaturing conditions. The resulting gels were either analyzed byCoomassie staining or transferred to polyvinyl difluoride (PVDF)membranes using the iBlot® (Life Technologies, Carlsbad, Calif., U.S.)system according to manufacturer's instructions. The resulting membraneswere probed under standard conditions using the following antibodies:rabbit polyclonal α-NWSHPQFEK (A00626, Genscript, Piscataway, N.J.,U.S.) which recognizes the polypeptide NWSHPQFEK also known as Streptag®II, mouse monoclonal α-Stx (mAb1 or anti-SLT-1A mAb1) (BEI NR-867 BEIResources, Manassas, Va., U.S.; cross reactive with Shiga toxin andShiga-like toxin 1 A subunits), rabbit polyclonal antibody α-SLT-1A(pAb1 or anti-SLT-1A pAb1) (Harlan Laboratories, Inc. Indianapolis,Ind., U.S., custom antibody production raised against the SLT-1A aminoacids 1-251), and rabbit polyclonal antibody α-SLT-1A (pAb2 oranti-SLT-1A pAb2) (Genscript, Piscataway, N.J., U.S., custom antibodyproduction), which was raised against the peptides RGIDPEEGRFNN andHGQDSVRVGR. The peptide sequence RGIDPEEGRFNN is located at amino acids55-66 in SLT-1A and StxA, spanning a predicted B cell epitope, and thepeptide sequence HGQDSVRVGR is located at 214-223 in SLT-1A and StxA,spanning a predicted B-cell epitope.

Membrane bound antibodies were detected using standard conditions and,when appropriate, using horseradish peroxidase (HRP) conjugatedsecondary antibodies (goat anti-rabbit-HRP or goat anti-mouse-HRP,Thermo Scientific, Rockford, Ill., U.S.). FIGS. 3-4 show images ofWestern blots with the lanes of the gels and/or membranes numbered andthe figure legends indicate by the same respective numbering which Shigatoxin effector polypeptide was a component of the cytotoxic proteinsample loaded into each lane. For each gel, the Coomassie stainingand/or anti-streptag II Western blot signal serve as total cytotoxicprotein loading controls. All the modified Shiga toxin effectorpolypeptides had reduced or abolished recognition by one or moreantibodies that can recognize wild-type SLT-1A indicating a reducedantigenicity and successful de-immunization. The result of the Westernblot analyses shown in FIGS. 3 and 4 are summarized in Table 13.

TABLE 13 Epitope Disruption Analysis by Western: Exemplary Shiga toxineffector polypeptides tested show reduced or abolished antibody bindingWestern Blot Result anti-SLT- anti-SLT- anti-SLT-Cytotoxic Protein comprising: 1A pAb1 1A pAb2 1A mAb1WT Shiga toxin effector region present present presentExemplary Shiga toxin effector polypeptide comprising a B-Cell epitoperegion disrupted with the T-cell epitope below: B-Cell T-Cell T-CellEpitope Epitope Epitope Region Position Embedded Disrupted  4-12ILRFSVAHK 1 reduced present abolished 43-51 NLVPMVATV 3 reduced presentabolished 44-52 GILGFVFTL 3 strongly reduced abolished reduced 53-61GILGFVFTL 4 reduced abolished abolished 53-61 DILGFVFTL 4 reducedabolished not tested 53-61 DILGFDFTL 4 reduced abolished not tested53-61 NLVPMVATV 4 strongly strongly abolished reduced reduced 104-112NLVPMVATV 5 present present abolished 180-188 GILDDVFTL 7 presentpresent abolished

Testing CD4+ T-Cell De-Immunization

Disruptions in predicted CD4+ T-cell epitope regions are tested forreductions in CD4+ T-cell immunogenicity using assays of human CD4+T-cell proliferation in the presence of exogenously administeredpolypeptides and assays of human CD4+ dendritic T-cell stimulation inthe presence of human monocytes treated with administered polypeptides.

T-cell proliferation assays known to the skilled worker are used to testthe effectiveness of CD4+ T-cell epitope de-immunization in exemplarytoxin effector polypeptides comprising T-cell epitopes embedded orinserted into predicted CD4+ T-cell epitopes. The T-cell proliferationassay of this example involves the labeling of CD4+ T-cells and thenmeasuring changes in proliferation using flow cytometric methods inresponse to the administration of different peptides derived from eithera polypeptide de-immunized using the methods of embedding or inserting aheterologous CD8+ T-cell epitope (e.g., SEQ ID NOs: 11-43) or areference polypeptide that does not have any heterologous T-cell epitopeassociated with it.

A series of overlapping peptides derived from a polypeptide aresynthesized and tested in the CFSE CD4+ T cell proliferation assay(ProImmune Inc., Sarasota, Fla., U.S). Human CD8+ T-cell depleted,peripheral blood mononuclear cells (PBMCs) labeled with CFSE arecultured with 5 μM of each peptide of interest for seven days in sixreplicate wells. Each assay plate includes a set of untreated controlwells. The assay also incorporates reference antigen controls,comprising synthetic peptides for known MHC class II antigens.

The CD8+ T-cell depleted, PBMCs that proliferate in response to anadministered peptide will show a reduction in CFSE fluorescenceintensity as measured directly by flow cytometry. For a naïve T-cellanalysis, the Percentage Stimulation above background is determined foreach stimulated sample, through comparison with results from anunstimulated sample, such as by ranking with regard to fluorescentsignal, as negative, dim, or high. Counts for the CD4+CFSE T-cell dimpopulation in each sample are expressed as a proportion of the totalCD4+ T-cell population. The replicate values are used to calculatePercentage Stimulation above Background (proportion of CD4+ T-cell CFSEdim cells with antigen stimulation, minus proportion of CD4+ T-cell CFSEdim cells without antigen stimulation). The mean and standard error ofthe mean are calculated from the replicate values. A result isconsidered “positive” if the Percentage Stimulation above background isgreater than 0.5% and also greater than twice the standard error abovebackground. To allow for comparison of peptides, a Response Index iscalculated. This index is based on multiplying the magnitude of response(Percentage Stimulation above background) for each peptide by the numberof responding donors (Percentage Antigenicity) for each peptide.

Determining Relative CD4+ T-Cell Immunogenicity

The relative CD4+ T-cell immunogenicity of exemplary, full-lengthpolypeptides of the invention is determined using the followingdendritic cell (DC) T-cell proliferation assay. This DC T-cell assaymeasures CD4+ T-cell responses to exogenously administered polypeptidesor proteins. The DC T-cell assay is performed using ProImmune's DC-Tassay service to determine the relative levels of CD4+ T-cell drivenimmunogenicity between polypeptides, proteins, and cell-targetedmolecules of the invention as compared to the starting parentalpolypeptides, proteins, or cell-targeted molecules which lack theaddition of any heterologous T-cell epitope. The DC T-cell assay of thisexample involves testing human dendritic cells for antigen presentationof peptides derived from the administered polypeptide, protein, orcell-targeted molecule samples.

Briefly, healthy human donor tissues are used to isolate typed samplesbased on high-resolution MHC Class II tissue-typing. A cohort of 20, 40or 50 donors is used. First, monocytes obtained from human donor PBMCsare cultured in a defined medium to generate immature dendritic cells.Then, the immature dendritic cells are stimulated with a well-definedcontrol antigen and induced into a more mature phenotype by furtherculture in a defined medium. Next, CD8+ T-cell depleted donor PBMCs fromthe same human donor sample are labeled with CFSE. The CFSE-labeled,CD8+ T-cell depleted PBMCs are then cultured with the antigen-primed,dendritic cells for seven days to allow for CD4+ dendritic cellstimulation, after which eight replicates for each sample are tested. Asnegative controls, each dendritic cell culture series also includes aset of untreated dendritic cells. For a positive control, the assayincorporates two well-defined reference antigens, each comprising afull-length protein.

To evaluate dendritic cell based immunogenicity, the frequency of donorcell responses is analyzed across the study cohort. Positive responsesin the assay are considered indicative of a potential in vivo CD4+T-cell response. A positive response, measured as a percentage ofstimulation above background, is defined as percentages greater than 0.5percent (%) in 2 or more independent donor samples. The strength ofpositive donor cell responses is determined by taking the meanpercentage stimulation above background obtained across accepted donorsfor each sample. A Response Index is calculated by multiplying the valueof the strength of response by the frequency of the donors responding todetermine levels of CD4+ T-cell immunogenicity for each sample. Inaddition, a Response index, representing the relative CD4+ T-cellimmunogenicity is determined by comparing the results from two samples,one comprising a CD8+ T cell epitope embedded in a predicted CD4+ T-cellepitope region and a second variant which lacks any disruption to thesame predicted CD4+ T-cell region to determine if the disruption reducesthe CD4+ T-cell response of human donor cells.

Testing De-Immunization Via Relative Immunogenicity In Vivo

The relative immunogenicity levels of Shiga toxin effector polypeptidesare tested for de-immunization using mammalian models of the humanimmune system. Mice are intravenously administered cytotoxic proteins orpolypeptides comprising either wild-type (WT) or de-immunized forms ofthe Shiga toxin effector polypeptide component 3 times per week for twoweeks or more. Blood samples are taken from the injected mice and testedby enzyme-linked immunosorbent assay (ELISA) for reactivity to thecytotoxic proteins and/or the Shiga toxin effector polypeptide. Reducedimmunogenic responses will be elicited in mice injected with thede-immunized Shiga toxin effector polypeptide, or compositionscomprising the same, as compared to mice injected only with thewild-type form of the Shiga toxin effector polypeptide, or compositioncomprising the same. The relatively reduced immunogenic response willindicate that the de-immunized Shiga toxin effector polypeptides arede-immunized with regard to having reduced immunogenic potential afteradministration to a mammal and allowing time for the mammal's immunesystem to respond.

In addition, diphtheria toxin effector polypeptides of the invention(e.g. SEQ ID NOs: 46-48) are tested for de-immunization using themethods of this example to verify the disruption of one or more B-cellepitope regions in each diphtheria toxin effector polypeptidescomprising an embedded or inserted T-cell epitope.

Example 4 Testing Cellular Internalization, Sub-Cellular Routing, andPresentation of an Embedded T-Cell Epitope on the Surfaces of TargetCells by Exemplary Shiga Toxin Effector Polypeptides of the Invention

In this example, the ability of exemplary cell-targeted proteins of theinvention, which each comprise an exemplary Shiga toxin effectorpolypeptide of the invention, to deliver T-cell epitopes to the MHCclass I pathway of target cells for presentation to the target cellsurface was investigated. In addition, cell-targeted proteins comprisingdiphtheria toxin effector polypeptides of the invention (e.g. SEQ IDNOs: 46-48) are tested using the methods of this example to verify theirability to deliver embedded T-cell epitopes to the MHC class Ipresentation system.

Using standard techniques known in the art, various exemplarycell-targeted proteins of the invention were made where each comprises acell-type-targeting region and a Shiga toxin effector polypeptide of theinvention (see e.g. WO2014164680 and WO2014164693). A cell-targetedprotein of the invention comprises both a Shiga toxin effectorpolypeptide of the invention and a cell-targeting binding region capableof exhibiting high-affinity binding to an extracellular targetbiomolecule physically-coupled to the surface of a specific celltype(s). The cell-targeted proteins of the invention are capable ofselectively targeting cells expressing the target biomolecule of theircell-targeting binding region and internalizing into these target cells.

A flow cytometry method was used to demonstrate delivery andextracellular display of a T-cell epitope (inserted or embedded in aShiga toxin effector region) in complex with MHC Class I molecules onthe surfaces of target cells. This flow cytometry method utilizessoluble human T-cell receptor (TCR) multimer reagents (Soluble T-CellAntigen Receptor STAR™ Multimer, Altor Bioscience Corp., Miramar, Fla.,U.S.), each with high-affinity binding to a different epitope-human HLAcomplex.

Each STAR™ TCR multimer reagent is derived from a specific T-cellreceptor and allows detection of a specific peptide-MHC complex based onthe ability of the chosen TCR to recognize a specific peptide presentedin the context of a particular MHC class I molecule. These TCR multimersare composed of recombinant human TCRs which have been biotinylated andmultimerized with streptavidin. The TCR multimers are labeled withphycoerythrin (PE). These TCR multimer reagents allow the detection ofspecific peptide-MHC Class I complexes presented on the surfaces ofhuman cells because each soluble TCR multimer type recognizes and stablybinds to a specific peptide-MHC complex under varied conditions (Zhu Xet al., J Immunol 176: 3223-32 (2006)). These TCR multimer reagentsallow the identification and quantitation by flow cytometry ofpeptide-MHC class I complexes present on the surfaces of cells.

The TCR CMV-pp65-PE STAR™ multimer reagent (Altor Bioscience Corp.,Miramar, Fla., U.S.) was used in this Example. MHC class I pathwaypresentation of the CMV C2 peptide (NLVPMVATV) by human cells expressingthe HLA-A2 can be detected with the TCR CMV-pp65-PE STAR™ multimerreagent which exhibits high affinity recognition of the CMV-pp65epitope-peptide (residues 495-503, NLVPMVATV) complexed to human HLA-A2and which is labeled with PE.

The target cells used in this Example were immortalized human cancercells available from the ATCC (Manassas Va., U.S.). Using standard flowcytometry methods known in the art, the target cells were confirmed toexpress on their cell surfaces both the HLA-A2 MHC-Class I molecule andthe extracellular target biomolecule of the cell-targeting moiety of theproteins used in this Example.

The target cells were treated with the exemplary cell-targeted proteinsof the invention, each comprising different Shiga toxin effectorpolypeptides comprising a T-cell epitope embedded into a predictedB-cell epitope region. One of each of the exemplary cell-targetedproteins of the invention tested in this Example comprised one of thefollowing Shiga toxin effector polypeptides: 43-51-C2 (SEQ ID NO: 13),53-61-C2(SEQ ID NO: 17), and 104-112-C2(SEQ ID NO: 18). Sets of targetcells were treated by exogenous administration of the differentexemplary cell-targeted proteins of the invention at concentrationssimilar to those used by others taking into account cell-type specificsensitivities to Shiga toxins (see e.g. Noakes K et al., FEBS Lett 453:95-9 (1999)). The treated cells were then incubated for six hours instandard conditions, including at 37° C. and an atmosphere with 5%carbon dioxide, to allow for intoxication mediated by a Shiga toxineffector region. Then the cells were washed with cell culture medium,re-suspended in fresh cell culture medium, and incubated for 20 hoursprior to staining with the TCR CMV-pp65-PE STAR™ multimer reagent.

As controls, sets of target cells were treated in three conditions: 1)without any treatment (“untreated”) meaning that no exogenous moleculeswere added, 2) with exogenously administered CMV C2 peptide (CMV-pp65,aa495-503: sequence NLVPMVATV, synthesized by BioSynthesis, Lewisville,Tex., U.S.), and 3) with exogenously administered CMV C2 peptide(NLVPMVATV, as above) combined with a Peptide Loading Enhancer (“PLE,”Altor Biosicence Corp., Miramar, Fla.). The C2 peptide combined with PLEtreatment allowed for exogenous peptide loading and served as a positivecontrol. Cells displaying the appropriate MHC class I haplotype can beforced to load the appropriate exogenously applied peptide from anextracellular space (i.e. in the absence of cellular internalization ofthe applied peptide) or in the presence of PLE, which is a mixture ofB2-microglobulin and other components.

After the treatments, all the sets of cells were washed and incubatedwith the TCR CMV-pp65-PE STAR multimer reagent for one hour on ice. Thecells were washed and the fluorescence of the samples was measured byflow cytometry using an Accuri™ C6 flow cytometer (BD Biosciences, SanJose, Calif., U.S.) to detect the presence of and quantify any TCRCMV-pp65-PE STAR™ multimer bound to cells in the population (sometimesreferred to herein as “staining”).

The results of the flow cytometric analysis of the sets of differentlytreated cells are shown in FIG. 5 and Table 14. The untreated controlwas used to identify the positive and negative cell populations byemploying a gate which results in less than 1% of cells from theuntreated control in the “positive” gate (representing backgroundsignal). The same gate was then applied to the other samples tocharacterize the positive population for each sample. In FIG. 5, theflow cytometry histograms are given with the counts (number of cells) onthe Y-axis and the relative fluorescent units (RFU) on the X-axis (logscale). The grey line in all histograms shows the profile of theuntreated cells and the black line shows the profile of treated cellsaccording to the treatment indicated. In Table 14, the percentage ofcells in a treatment set which stained positive for theC2-epitope-peptide-HLA-A2 complex is given. Positive cells in this assaywere cells which were bound by the TCR-CMV-pp65-PE STAR reagent andcounted in the positive gate described above. Table 14 also shows foreach set the corresponding indexed, mean, fluorescent intensity (“iMFI,”the fluorescence of the positive population multiplied by the percentpositive) in RFU.

TABLE 14 Flow Cytometry Results for Exemplary Cell-targeted proteins ofthe invention: Peptide-epitope C2-MHC class I complexes detected on thesurfaces of intoxicated, target cells TCR CMV-pp65-PE Flow CytometryTarget cell treatment: exogenously Percentage of administeredmolecule(s) Positive Cells iMFI (RFU) Untreated 0.96% 20 Cell-targetedprotein with Shiga toxin  7.6% 113 effector region 43-51-C2Cell-targeted protein with Shiga toxin  4.5% 64 effector region 53-61-C2Cell-targeted protein with Shiga toxin  6.7% 89 effector region104-112-C2 C2 peptide only 0.95% 19 C2 peptide and PLE 36.7% 728

Cells administered with exogenous protein comprising 43-51-C2, 53-61-C2,and 104-112-C2 showed a positive signal for cell-surface,C2-peptide-HLA-A2 complexes on 7.6%, 4.5%, and 6.7% of the cells intheir population, respectively. In contrast, cell populations that were“untreated” and treated with “C2 peptide only” contained less than 1%positive cells (0.96 and 0.95 percent, respectively). Due to processingefficiency and kinetics, which were not measured, it is possible thatthe percent of presented C2-peptide-HLA-A2 complex detected at a singletimepoint in a “cell-targeted protein” treatment sample may notaccurately reflect the maximum presentation possible by these exemplarycell-targeted proteins of the invention.

The positive control “C2 peptide and PLE” population contained 36.7%positive cells; however, the peptide can only be loaded onto the surfacefrom an extracellular space (“exogenously”) and in the presence of PLEas shown by comparing with the “C2 peptide only” results which had asimilar background staining level (0.95%) as the untreated control.

The detection of the exogenously administered, embedded T-cell epitopeC2 complexed with human MHC Class I molecules (C2epitope-peptide/HLA-A2) on the cell surface of intoxicated target cellsdemonstrated that cell-targeted proteins comprising the exemplary Shigatoxin effector regions 43-51-C2, 53-61-C2, or 104-112-C2 were capable ofentering target cells, performing sufficient sub-cellular routing, anddelivering enough embedded T-cell epitope to the MHC class I pathway forsurface presentation on the target cell surface.

Example 5 Testing Cytotoxic T-Cell Mediated Cytolysis of IntoxicatedTarget Cells and Other Immune Responses Triggered by MHC Class IPresentation of T-Cell Epitopes Delivered by Proteins of the PresentInvention

In this example, standard assays known in the art are used toinvestigate the functional consequences of target cells' MHC class Ipresentation of T-cell epitopes delivered by exemplary cell-targetedproteins of the invention. The functional consequences to investigateinclude CTL activation, CTL mediated target cell killing, and CTLcytokine release by CTLs.

A CTL-based cytotoxicity assay is used to assess the consequences ofepitope presentation. The assay involves tissue-cultured target cellsand T-cells. Target cells are intoxicated as described in Example 4.Briefly, target cells are incubated for six hours in standard conditionswith different exogenously administered proteins, where certain proteinscomprise a Shiga toxin effector polypeptide of the invention or adiphtheria toxin effector polypeptide of the invention. Next, CTLs areadded to the intoxicated target cells and incubated to allow for theT-cells to recognize and bind any target-cells displayingepitope-peptide/MHC class I complexes. Then certain functionalconsequences are investigated using standard methods known to theskilled worker, including CTL binding to target cells, target cellkilling by CTL-mediated cytolysis, and the release of cytokines, such asinterferon gamma or interleukins by ELISA or ELIspot.

The activation of CTLs by target cells displaying epitope-peptide/MHCclass I complexes is quantified using commercially available CTLresponse assays, e.g. CytoTox96® non-radioactive assays (Promega,Madison, Wis., U.S.), Granzyme B ELISpot assays (Mabtech, Inc.,Cincinnati, Ohio, U.S.), caspase activity assays, and LAMP-1translocation flow cytometric assays. To specifically monitorCTL-mediated killing of target cells, carboxyfluorescein succinimidylester (CFSE) is used to target-cells for in vitro and in vivoinvestigation as described in the art (see e.g. Durward M et al., J VisExp 45 pii 2250 (2010)).

Example 6 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide and aBinding Region Specific to CD20 (αCD20 Fused with SLT-1A)

In this example, a T-cell hyper-immunized and B-cell/CD4+ T-cellde-immunized Shiga toxin effector region is derived from the A subunitof Shiga-like Toxin 1 (SLT-1A) as described above. Animmunoglobulin-type binding region αCD20-antigen is derived from animmunoglobulin-type domain recognizing human CD20 (see e.g. Haisma etal., Blood 92: 184-90 (1999); Geng S et al., Cell Mol Immunol 3: 439-43(2006); Olafesn T et al., Protein Eng Des Sel 23: 243-9 (2010)), whichcomprises an immunoglobulin-type binding region capable of binding anextracellular part of CD20. CD20 is expressed on multiple cancer celltypes, such as, e.g., B-cell lymphoma cells, hairy cell leukemia cells,B-cell chronic lymphocytic leukemia cells, and melanoma cells. Inaddition, CD20 is an attractive target for therapeutics to treat certainautoimmune diseases, disorders, and conditions involving overactiveB-cells.

Construction, Production, and Purification of the Cytotoxic ProteinSLT-1A::αCD20

The immunoglobulin-type binding region αCD20 and Shiga toxin effectorregion (such as, e.g., SEQ ID NOs: 11-43) are linked together. Forexample, a fusion protein is produced by expressing a polynucleotideencoding the αCD20-antigen-binding protein SLT-1A::αCD20 (see, e.g., SEQID NOs: 49, 50, and 51). Expression of the SLT-1A::αCD20 cytotoxicprotein is accomplished using either bacterial and/or cell-free, proteintranslation systems as described in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic ProteinSLT-1A::αCD20

The binding characteristics, the maximum specific binding (B_(max)) andequilibrium binding constants (K_(D)), of the cytotoxic protein of thisexample for CD20+ cells and CD20− cells is determined byfluorescence-based, flow-cytometry. The B_(max) for SLT-1A::αCD20 toCD20+ cells is measured to be approximately 50,000-200,000 MFI with aK_(D) within the range of 0.01-100 nM, whereas there is no significantbinding to CD20− cells in this assay.

The ribosome inactivation abilities of the SLT-1A::αCD20 cytotoxicprotein is determined in a cell-free, in vitro protein translation asdescribed above in the previous examples. The inhibitory effect of thecytotoxic protein of this example on cell-free protein synthesis issignificant. The IC₅₀ of SLT-1A::αCD20 on protein synthesis in thiscell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic Protein SLT-1A::αCD20Using a CD20+ Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A::αCD20 are determined by thegeneral cell-kill assay as described above in the previous examplesusing CD20+ cells. In addition, the selective cytotoxicitycharacteristics of SLT-1A::αCD20 are determined by the same generalcell-kill assay using CD20− cells as a comparison to the CD20+ cells.The CD₅₀ of the cytotoxic protein of this example is approximately0.01-100 nM for CD20+ cells depending on the cell line. The CD₅₀ of thecytotoxic protein is approximately 10-10,000 fold greater (lesscytotoxic) for cells not expressing CD20 on a cellular surface ascompared to cells which do express CD20 on a cellular surface. Inaddition, the cytotoxicity of SLT-1A::αCD20 is investigated for bothdirect cytotoxicity and indirect cytotoxicity by T-cell epitope deliveryand presentation leading to CTL-mediated cytotoxicity.

Determining the In Vivo Effects of the Cytotoxic Protein SLT-1A::αCD20Using Animal Models

Animal models are used to determine the in vivo effects of the cytotoxicprotein SLT-1A::αCD20 on neoplastic cells. Various mice strains are usedto test the effect of the cytotoxic protein after intravenousadministration on xenograft tumors in mice resulting from the injectioninto those mice of human neoplastic cells which express CD20 on theircell surfaces. Cell killing is investigated for both direct cytotoxicityand indirect cytotoxicity by T-cell epitope delivery and presentationleading to CTL-mediated cytotoxicity.

Example 7 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide and aBinding Region Specific to HER2 (“αHER2-V_(H)H Fused with SLT-1A”)

In this example, the CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cellde-immunized Shiga toxin effector region is derived from the A subunitof Shiga-like Toxin 1 (SLT-1A) as described above. Theimmunoglobulin-type binding region is αHER2 V_(H)H derived from asingle-domain variable region of the camelid antibody (V_(H)H) protein5F7, as described in U.S. Patent Application Publication 2011/0059090.

Construction, Production, and Purification of the Cytotoxic Protein“αHER2-V_(H)H Fused with SLT-1A”

The immunoglobulin-type binding region and Shiga toxin effector regionare linked together to form a fused protein (see, e.g., SEQ ID NOs: 52,53, and 54). In this example, a polynucleotide encoding the αHER2-V_(H)Hvariable region derived from protein 5F7 may be cloned in frame with apolynucleotide encoding a linker known in the art and in frame with apolynucleotide encoding the Shiga toxin effector region comprising aminoacids of SEQ ID NOs: 11-43. Variants of “αHER2-V_(H)H fused with SLT-1A”cytotoxic proteins are created such that the binding region isoptionally located adjacent to the amino-terminus of the Shiga toxineffector region and optionally comprises a carboxy-terminal endoplasmicreticulum signal motif of the KDEL family. Expression of the“αHER2-V_(H)H fused with SLT-1A” cytotoxic protein variants isaccomplished using either bacterial and/or cell-free, proteintranslation systems as described in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic Protein“αHER2-V_(H)H Fused with SLT-1A”

The binding characteristics of the cytotoxic protein of this example forHER2+ cells and HER2− cells is determined by a fluorescence-based,flow-cytometry. The B_(max) for “αHER2-V_(H)H fused with SLT-1A”variants to HER2+ cells is measured to be approximately 50,000-200,000MFI with a K_(D) within the range of 0.01-100 nM, whereas there is nosignificant binding to HER2− cells in this assay.

The ribosome inactivation abilities of the “αHER2-V_(H)H fused withSLT-1A” cytotoxic proteins are determined in a cell-free, in vitroprotein translation as described above in the previous examples. Theinhibitory effect of the cytotoxic protein of this example on cell-freeprotein synthesis is significant. The IC₅₀ of “αHER2-V_(H)H fused withSLT-1A” variants on protein synthesis in this cell-free assay isapproximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic Protein “αHER2-V_(H)HFused with SLT-1A” Using a HER2+ Cell-Kill Assay

The cytotoxicity characteristics of “αHER2-V_(H)H fused with SLT-1A”variants are determined by the general cell-kill assay as describedabove in the previous examples using HER2+ cells. In addition, theselective cytotoxicity characteristics of “αHER2-V_(H)H fused withSLT-1A” are determined by the same general cell-kill assay using HER2−cells as a comparison to the HER2+ cells. The CD₅₀ of the cytotoxicprotein of this example is approximately 0.01-100 nM for HER2+ cellsdepending on the cell line. The CD₅₀ of the cytotoxic protein isapproximately 10-10,000 fold greater (less cytotoxic) for cells notexpressing HER2 on a cellular surface as compared to cells which doexpress HER2 on a cellular surface. In addition, the cytotoxicity ofαHER2-V_(H)H fused with SLT-1A is investigated for both directcytotoxicity and indirect cytotoxicity by T-cell epitope delivery andpresentation leading to CTL-mediated cytotoxicity.

Determining the In Vivo Effects of the Cytotoxic Protein αHER2-V_(H)HFused with SLT-1A Using Animal Models

Animal models are used to determine the in vivo effects of the cytotoxicprotein αHER2-V_(H)H fused with SLT-1A on neoplastic cells. Various micestrains are used to test the effect of the cytotoxic protein afterintravenous administration on xenograft tumors in mice resulting fromthe injection into those mice of human neoplastic cells which expressHER2 on their cell surfaces. Cell killing is investigated for bothdirect cytotoxicity and indirect cytotoxicity by T-cell epitope deliveryand presentation leading to CTL-mediated cytotoxicity.

Example 8 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide and aBinding Region Derived from the Antibody αEpstein-Barr-Antigen

In this example, the CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cellde-immunized Shiga toxin effector region is a de-immunized Shiga toxineffector polypeptide derived from the A subunit of Shiga-like Toxin 1(SLT-1A) as described above. An immunoglobulin-type binding regionaEpstein-Barr-antigen is derived from a monoclonal antibody against anEpstein Barr antigen (Fang C et al., J Immunol Methods 287: 21-30(2004)), which comprises an immunoglobulin-type binding region capableof binding a human cell infected by the Epstein-Barr virus or atransformed cell expressing an Epstein-Barr antigen. The Epstein-Barrantigen is expressed on multiple cell types, such as cells infected byan Epstein-Barr virus and cancer cells (e.g. lymphoma and nasphamygealcancer cells). In addition, Epstein-Barr infection is associated withother diseases, e.g., multiple sclerosis.

Construction, Production, and Purification of the Cytotoxic ProteinSLT-1A::αEpsteinBarr::KDEL

The immunoglobulin-type binding region aEpstein-Barr-antigen and Shigatoxin effector region are linked together, and a carboxy-terminal KDELis added to form a protein. For example, a fusion protein is produced byexpressing a polynucleotide encoding the aEpstein-Barr-antigen-bindingprotein SLT-1A::αEpsteinBarr::KDEL. Expression of theSLT-1A::αEpsteinBarr::KDEL cytotoxic protein is accomplished usingeither bacterial and/or cell-free, protein translation systems asdescribed in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic ProteinSLT-1A::αEpsteinBarr::KDEL

The binding characteristics of the cytotoxic protein of this example forEpstein-Barr antigen positive cells and Epstein-Barr antigen negativecells is determined by fluorescence-based, flow-cytometry. The B_(max)for SLT-1A::αEpsteinBarr::KDEL to Epstein-Barr antigen positive cells ismeasured to be approximately 50,000-200,000 MFI with a K_(D) within therange of 0.01-100 nM, whereas there is no significant binding toEpstein-Barr antigen negative cells in this assay.

The ribosome inactivation abilities of the SLT-1A::αEpsteinBarr::KDELcytotoxic protein is determined in a cell-free, in vitro proteintranslation as described above in the previous examples. The inhibitoryeffect of the cytotoxic protein of this example on cell-free proteinsynthesis is significant. The IC₅₀ of SLT-1A::αEpsteinBarr::KDEL onprotein synthesis in this cell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic ProteinSLT-1A::αEpsteinBarr::KDEL Using a Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A::αEpsteinBarr::KDEL aredetermined by the general cell-kill assay as described above in theprevious examples using Epstein-Barr antigen positive cells. Inaddition, the selective cytotoxicity characteristics ofSLT-1A::αEpsteinBarr::KDEL are determined by the same general cell-killassay using Epstein-Barr antigen negative cells as a comparison to theEpstein-Barr antigen positive cells. The CD₅₀ of the cytotoxic proteinof this example is approximately 0.01-100 nM for Epstein-Barr antigenpositive cells depending on the cell line. The CD₅₀ of the cytotoxicprotein is approximately 10-10,000 fold greater (less cytotoxic) forcells not expressing the Epstein-Barr antigen on a cellular surface ascompared to cells which do express the Epstein-Barr antigen on acellular surface. In addition, the cytotoxicity ofSLT-1A::αEpsteinBarr::KDEL is investigated for both direct cytotoxicityand indirect cytotoxicity by T-cell epitope delivery and presentationleading to CTL-mediated cytotoxicity.

Determining the In Vivo Effects of the Cytotoxic ProteinSLT-1A::αEpsteinBarr::KDEL Using Animal Models

Animal models are used to determine the in vivo effects of the cytotoxicprotein SLT-1A::αEpsteinBarr::KDEL on neoplastic cells. Various micestrains are used to test the effect of the cytotoxic protein afterintravenous administration on xenograft tumors in mice resulting fromthe injection into those mice of human neoplastic cells which expressEpstein-Barr antigens on their cell surfaces. Cell killing isinvestigated for both direct cytotoxicity and indirect cytotoxicity byT-cell epitope delivery and presentation leading to CTL-mediatedcytotoxicity.

Example 9 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide and aBinding Region Derived from the Antibody αLeishmania-Antigen

In this example, the Shiga toxin effector region is a CD8+ T-cellhyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effectorpolypeptide derived from the A subunit of Shiga-like Toxin 1 (SLT-1A) asdescribed above. An immunoglobulin-type binding regionaLeishmania-antigen is derived from an antibody generated, usingtechniques known in the art, to a cell-surface Leishmania antigenpresent on human cells harboring an intracellular trypanosomatidprotozoa (see Silveira T et al., Int J Parasitol 31: 1451-8 (2001);Kenner J et al., J Cutan Pathol 26: 130-6 (1999); Berman J and Dwyer,Clin Exp Immunol 44: 342-348 (1981)).

Construction, Production, and Purification of the Cytotoxic ProteinSLT-1A::αLeishmania::KDEL

The immunoglobulin-type binding region α-Leishmania-antigen and Shigatoxin effector region are linked together, and a carboxy-terminal KDELis added to form a protein. For example, a fusion protein is produced byexpressing a polynucleotide encoding the Leishmania-antigen-bindingprotein SLT-1A::αLeishmania::KDEL. Expression of theSLT-1A::αLeishmania::KDEL cytotoxic protein is accomplished using eitherbacterial and/or cell-free, protein translation systems as described inthe previous examples.

Determining the In Vitro Characteristics of the Cytotoxic ProteinSLT-1A::αLeishmania::KDEL

The binding characteristics of the cytotoxic protein of this example forLeishmania antigen positive cells and Leishmania antigen negative cellsis determined by fluorescence-based, flow-cytometry. The B_(max) forSLT-1A::αLeishmania::KDEL to Leishmania antigen positive cells ismeasured to be approximately 50,000-200,000 MFI with a K_(D) within therange of 0.01-100 nM, whereas there is no significant binding toLeishmania antigen negative cells in this assay.

The ribosome inactivation abilities of the SLT-1A::αLeishmania::KDELcytotoxic protein is determined in a cell-free, in vitro proteintranslation as described above in the previous examples. The inhibitoryeffect of the cytotoxic protein of this example on cell-free proteinsynthesis is significant. The IC₅₀ of SLT-1A::αLeishmania::KDEL onprotein synthesis in this cell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic ProteinSLT-1A::αLeishmania::KDEL Using a Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A::αLeishmania::KDEL aredetermined by the general cell-kill assay as described above in theprevious examples using Leishmania antigen positive cells. In addition,the selective cytotoxicity characteristics of SLT-1A::αLeishmania::KDELare determined by the same general cell-kill assay using Leishmaniaantigen negative cells as a comparison to the Leishmania antigenpositive cells. The CD₅₀ of the cytotoxic protein of this example isapproximately 0.01-100 nM for Leishmania antigen positive cellsdepending on the cell line. The CD₅₀ of the cytotoxic protein isapproximately 10-10,000 fold greater (less cytotoxic) for cells notexpressing the Leishmania antigen on a cellular surface as compared tocells which do express the Leishmania antigen on a cellular surface. Inaddition, the cytotoxicity of SLT-1A::αLeishmania::KDEL is investigatedfor both direct cytotoxicity and indirect cytotoxicity by T-cell epitopedelivery and presentation leading to CTL-mediated cytotoxicity.

Example 10 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide and aBinding Region Derived from an Immunoglobulin-Type Binding RegionαNeurotensin-Receptor

In this example, the Shiga toxin effector region is a CD8+ T-cellhyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effectorpolypeptide derived from the A subunit of Shiga-like Toxin 1 (SLT-1A) asdescribed above. An immunoglobulin-type binding regionαNeurotensin-Receptor is derived from the DARPin™ (GenBank Accession:2P2C_R) or a monoclonal antibody (Ovigne J et al., Neuropeptides 32:247-56 (1998)) which binds the human neurotensin receptor. Theneurotensin receptor is expressed by various cancer cells, such asbreast cancer, colon cancer, lung cancer, melanoma, and pancreaticcancer cells.

Construction, Production, and Purification of the Cytotoxic ProteinSLT-1A::αNeurotensinR::KDEL

The immunoglobulin-type binding region αNeurotensinR and Shiga toxineffector region are linked together, and a carboxy-terminal KDEL isadded to form a protein. For example, a fusion protein is produced byexpressing a polynucleotide encoding the neurotensin-receptor-bindingprotein SLT-1A::αNeurotensinR::KDEL. Expression of theSLT-1A::αNeurotensinR::KDEL cytotoxic protein is accomplished usingeither bacterial and/or cell-free, protein translation systems asdescribed in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic ProteinSLT-1A::αNeurotensinR::KDEL

The binding characteristics of the cytotoxic protein of this example forneurotensin receptor positive cells and neurotensin receptor negativecells is determined by fluorescence-based, flow-cytometry. The B_(max)for SLT-1A::αNeurotensinR::KDEL to neurotensin receptor positive cellsis measured to be approximately 50,000-200,000 MFI with a K_(D) withinthe range of 0.01-100 nM, whereas there is no significant binding toneurotensin receptor negative cells in this assay.

The ribosome inactivation abilities of the SLT-1A::αNeurotensinR::KDELcytotoxic protein is determined in a cell-free, in vitro proteintranslation as described above in the previous examples. The inhibitoryeffect of the cytotoxic protein of this example on cell-free proteinsynthesis is significant. The IC₅₀ of SLT-1A::αNeurotensinR::KDEL onprotein synthesis in this cell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic ProteinSLT-1A::αNeurotensinR::KDEL Using a Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A::αNeurotensinR::KDEL aredetermined by the general cell-kill assay as described above in theprevious examples using neurotensin receptor positive cells. Inaddition, the selective cytotoxicity characteristics ofSLT-1A::αNeurotensinR::KDEL are determined by the same general cell-killassay using neurotensin receptor negative cells as a comparison to theneurotensin receptor positive cells. The CD₅₀ of the cytotoxic proteinof this example is approximately 0.01-100 nM for neurotensin receptorpositive cells depending on the cell line. The CD₅₀ of the cytotoxicprotein is approximately 10-10,000 fold greater (less cytotoxic) forcells not expressing neurotensin receptor on a cellular surface ascompared to cells which do express neurotensin receptor on a cellularsurface. In addition, the cytotoxicity of SLT-1A::αNeurotensinR::KDEL isinvestigated for both direct cytotoxicity and indirect cytotoxicity byT-cell epitope delivery and presentation leading to CTL-mediatedcytotoxicity.

Determining the In Vivo Effects of the Cytotoxic ProteinSLT-1A::αNeurotensinR::KDEL Using Animal Models

Animal models are used to determine the in vivo effects of the cytotoxicprotein SLT-1A::αNeurotensinR::KDEL on neoplastic cells. Various micestrains are used to test the effect of the cytotoxic protein afterintravenous administration on xenograft tumors in mice resulting fromthe injection into those mice of human neoplastic cells which expressneurotensin receptors on their cell surfaces. Cell killing isinvestigated for both direct cytotoxicity and indirect cytotoxicity byT-cell epitope delivery and presentation leading to CTL-mediatedcytotoxicity.

Example 11 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide and aBinding Region Derived from an Immunoglobulin-Type Binding Region αEGFR

In this example, the Shiga toxin effector region is CD8+ T-cellhyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effectorpolypeptide derived from the A subunit of Shiga-like Toxin 1 (SLT-1A).The binding region αEGFR is derived from the AdNectin™ (GenBankAccession: 3QWQ_B), the Affibody™ (GenBank Accession: 2KZI_A; U.S. Pat.No. 8,598,113), or an antibody, all of which bind one or more humanepidermal growth factor receptors. The expression of epidermal growthfactor receptors are associated with human cancer cells, such as, e.g.,lung cancer cells, breast cancer cells, and colon cancer cells.

Construction, Production, and Purification of the Cytotoxic ProteinSLT-1A::αEGFR::KDEL

The immunoglobulin-type binding region αEGFR and Shiga toxin effectorregion are linked together, and a carboxy-terminal KDEL is added to forma protein. For example, a fusion protein is produced by expressing apolynucleotide encoding the EGFR binding protein SLT-1A::αEGFR::KDEL.Expression of the SLT-1A::αEGFR::KDEL cytotoxic protein is accomplishedusing either bacterial and/or cell-free, protein translation systems asdescribed in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic ProteinSLT-1A::αEGFR::KDEL

The binding characteristics of the cytotoxic protein of this example forEGFR+ cells and EGFR− cells is determined by fluorescence-based,flow-cytometry. The B_(max) for SLT-1A::αEGFR::KDEL to EGFR+ cells ismeasured to be approximately 50,000-200,000 MFI with a K_(D) within therange of 0.01-100 nM, whereas there is no significant binding to EGFR−cells in this assay.

The ribosome inactivation abilities of the SLT-1A::αEGFR::KDEL cytotoxicprotein is determined in a cell-free, in vitro protein translation asdescribed above in the previous examples. The inhibitory effect of thecytotoxic protein of this example on cell-free protein synthesis issignificant. The IC₅₀ of SLT-1A::αEGFR::KDEL on protein synthesis inthis cell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic ProteinSLT-1A::αEGFR::KDEL Using a Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A::αEGFR::KDEL are determinedby the general cell-kill assay as described above in the previousexamples using EGFR+ cells. In addition, the selective cytotoxicitycharacteristics of SLT-1A::αEGFR::KDEL are determined by the samegeneral cell-kill assay using EGFR− cells as a comparison to theLeishmania antigen positive cells. The CD₅₀ of the cytotoxic protein ofthis example is approximately 0.01-100 nM for EGFR+ cells depending onthe cell line. The CD₅₀ of the cytotoxic protein is approximately10-10,000 fold greater (less cytotoxic) for cells not expressing EGFR ona cellular surface as compared to cells which do express EGFR on acellular surface. In addition, the cytotoxicity of SLT-1A::αEGFR::KDELis investigated for both direct cytotoxicity and indirect cytotoxicityby T-cell epitope delivery and presentation leading to CTL-mediatedcytotoxicity.

Determining the In Vivo Effects of the Cytotoxic ProteinSLT-1A::αEGFR::KDEL Using Animal Models

Animal models are used to determine the in vivo effects of the cytotoxicprotein SLT-1A::αEGFR::KDEL on neoplastic cells. Various mice strainsare used to test the effect of the cytotoxic protein after intravenousadministration on xenograft tumors in mice resulting from the injectioninto those mice of human neoplastic cells which express EGFR(s) on theircell surfaces. Cell killing is investigated for both direct cytotoxicityand indirect cytotoxicity by T-cell epitope delivery and presentationleading to CTL-mediated cytotoxicity.

Example 12 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide and aBinding Region Derived from the Antibody αCCR5

In this example, the Shiga toxin effector region is a CD8+ T-cellhyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effectorpolypeptide derived from the A subunit of Shiga-like Toxin 1 (SLT-1A).An immunoglobulin-type binding region αCCR5 is derived from a monoclonalantibody against human CCR5 (CD195) (Bernstone L et al., Hybridoma 31:7-19 (2012)). CCR5 is predominantly expressed on T-cells, macrophages,dendritic cells, and microglia. In addition, CCR5 plays a role in thepathogenesis and spread of the Human Immunodeficiency Virus (HIV).

Construction, Production, and Purification of the Cytotoxic ProteinSLT-1A::αCCR5::KDEL

The immunoglobulin-type binding region αCCR5 and Shiga toxin effectorregion are linked together, and a carboxy-terminal KDEL is added to forma protein. For example, a fusion protein is produced by expressing apolynucleotide encoding the αCCR5-binding protein SLT-1A::αCCR5::KDEL.Expression of the SLT-1A::αCCR5::KDEL cytotoxic protein is accomplishedusing either bacterial and/or cell-free, protein translation systems asdescribed in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic ProteinSLT-1A::αCCR5

The binding characteristics of the cytotoxic protein of this example forCCR5+ cells and CCR5− cells is determined by fluorescence-based,flow-cytometry. The B_(max) for SLT-1A::αCCR5::KDEL to CCR5+ positivecells is measured to be approximately 50,000-200,000 MFI with a K_(D)within the range of 0.01-100 nM, whereas there is no significant bindingto CCR5− cells in this assay.

The ribosome inactivation abilities of the SLT-1A::αCCR5::KDEL cytotoxicprotein is determined in a cell-free, in vitro protein translation asdescribed above in the previous examples. The inhibitory effect of thecytotoxic protein of this example on cell-free protein synthesis issignificant. The IC₅₀ of SLT-1A::αCCR5::KDEL on protein synthesis inthis cell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic ProteinSLT-1A::αCCR5::KDEL Using a Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A::αCCR5::KDEL are determinedby the general cell-kill assay as described above in the previousexamples using CCR5+ cells. In addition, the selective cytotoxicitycharacteristics of SLT-1A::αCCR5::KDEL are determined by the samegeneral cell-kill assay using CCR5− cells as a comparison to the CCR5+cells. The CD₅₀ of the cytotoxic protein of this example isapproximately 0.01-100 nM for CCR5+ cells depending on the cell line.The CD₅₀ of the cytotoxic protein is approximately 10-10,000 foldgreater (less cytotoxic) for cells not expressing CCR5 on a cellularsurface as compared to cells which do express CCR5 on a cellularsurface. In addition, the cytotoxicity of SLT-1A::αCCR5::KDEL isinvestigated for both direct cytotoxicity and indirect cytotoxicity byT-cell epitope delivery and presentation leading to CTL-mediatedcytotoxicity.

Determining the In Vivo Effects of the Cytotoxic ProteinSLT-1A::αCCR5::KDEL Using Animal Models

Animal models are used to determine the in vivo effects of the cytotoxicprotein SLT-1A::αCCR5::KDEL on depleting T-cells from donor materials(see Tsirigotis P et al., Immunotherapy 4: 407-24 (2012)). Non-humanprimates are used to determine in vivo effects of SLT-1A::αCCR5. Graftversus host disease is analyzed in rhesus macaques after kidneytransplantation when the donated organs are pretreated withSLT-1A::αCCR5::KDEL (see Weaver T et al., Nat Med 15: 746-9 (2009)). Invivo depletion of peripheral blood T lymphocytes in cynomolgus primatesis observed after parenteral administration of different doses ofSLT-1A::αCCR5::KDEL. Cell killing is investigated for both directcytotoxicity and indirect cytotoxicity by T-cell epitope delivery andpresentation leading to CTL-mediated cytotoxicity. The use ofSLT-1A::αCCR5::KDEL to block HIV infection is tested by giving an acutedose of SLT-1A::αCCR5::KDEL to non-human primates in order to severelydeplete circulating T-cells upon exposure to a simian immunodeficiencyvirus (SIV) (see Sellier P et al., PLoS One 5: e10570 (2010)).

Example 13 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide and aBinding Region Derived from an Anti-Env Immunoglobulin Domain

In this example, the Shiga toxin effector region is a CD8+ T-cellhyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effectorpolypeptide derived from the A subunit of Shiga toxin (StxA). Animmunoglobulin-type binding region αEnv is derived from existingantibodies that bind HIV envelope glycoprotein (Env), such as GP41,GP120, GP140, or GP160 (see e.g. Chen W et al., J Mol Bio 382: 779-89(2008); Chen W et al., Expert Opin Biol Ther 13: 657-71 (2013); van denKerkhof T et al., Retrovirology 10: 102 (2013)) or from antibodiesgenerated using standard techniques (see Prabakaran et al., FrontMicrobiol 3: 277 (2012)). Envs are HIV surface proteins that are alsodisplayed on the cell surfaces of HIV-infected cells during HIVreplication. Although Envs are expressed in infected cells predominantlyin endosomal compartments, sufficient amounts of Envs could be presenton a cell surface to be targeted by a highly potent cytotoxic,cell-targeted protein of the invention. In addition, Env-targetingcytotoxic proteins might bind HIV virions and enter newly infected cellsduring the fusion of virions with a host cell.

Because HIV displays a high rate of mutation, it is preferable to use animmunoglobulin domain that binds a functional constrained part of anEnv, such as shown by broadly neutralizing antibodies that bind Envsfrom multiple strains of HIV (van den Kerkhof T et al., Retrovirology10: 102 (2013)). Because the Envs present on an infected cell's surfaceare believed to present sterically restricted epitopes (Chen W et al., JVirol 88: 1125-39 (2014)), it is preferable to use smaller than 100 kDand ideally smaller than 25 kD, such as sdAbs or V_(H)H domains.

Construction, Production, and Purification of the Cytotoxic ProteinSLT-1A::αEnv::KDEL

The immunoglobulin-type binding region αEnv and Shiga toxin effectorregion are linked together, and a carboxy-terminal KDEL is added to forma cytotoxic protein. For example, a fusion protein is produced byexpressing a polynucleotide encoding the αEnv-binding proteinSLT-1A::αEnv::KDEL. Expression of the SLT-1A::αEnv::KDEL cytotoxicprotein is accomplished using either bacterial and/or cell-free, proteintranslation systems as described in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic ProteinSLT-1A::αEnv::KDEL

The binding characteristics of the cytotoxic protein of this example forEnv+ cells and Env− cells is determined by fluorescence-based,flow-cytometry assay. The B_(max) for SLT-1A::αEnv::KDEL to Env+positive cells is measured to be approximately 50,000-200,000 MFI with aK_(D) within the range of 0.01-100 nM, whereas there is no significantbinding to Env− cells in this assay.

The ribosome inactivation abilities of the SLT-1A::αEnv::KDEL cytotoxicprotein is determined in a cell-free, in vitro protein translation asdescribed above in the previous examples. The inhibitory effect of thecytotoxic protein of this example on cell-free protein synthesis issignificant. The IC₅₀ of SLT-1A::αEnv::KDEL on protein synthesis in thiscell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic Protein SLT-1A::αEnv::KDELUsing a Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A::αEnv::KDEL are determined bythe general cell-kill assay as described above in the previous examplesusing Env+ cells. In addition, the selective cytotoxicitycharacteristics of SLT-1A::αEnv::KDEL are determined by the same generalcell-kill assay using Env-cells as a comparison to the Env+ cells. TheCD₅₀ of the cytotoxic protein of this example is approximately 0.01-100nM for Env+ cells depending on the cell line and/or the HIV strain usedto infect the cells to make them Env+. The CD₅₀ of the cytotoxic proteinis approximately 10-10,000 fold greater (less cytotoxic) for cells notexpressing Env on a cellular surface as compared to cells which doexpress Env on a cellular surface. In addition, the cytotoxicity ofSLT-1A::αEnv::KDEL is investigated for both direct cytotoxicity andindirect cytotoxicity by T-cell epitope delivery and presentationleading to CTL-mediated cytotoxicity.

Determining the In Vivo Effects of the Cytotoxic Protein SLT-1A::Env::KDEL Using Animal Models

The use of SLT-1A::αEnv::KDEL to inhibit HIV infection is tested byadministering SLT-1A::αEnv::KDEL to simian immunodeficiency virus (SIV)infected non-human primates (see Sellier P et al., PLoS One 5: e10570(2010)). Cell killing is investigated for both direct cytotoxicity andindirect cytotoxicity by T-cell epitope delivery and presentationleading to CTL-mediated cytotoxicity.

Example 14 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Shiga Toxin Effector Polypeptide and aBinding Region Derived from the Antibody αUL18

In this example, the Shiga toxin effector region is a CD8+ T-cellhyper-immunized and B-cell/CD4+ T-cell de-immunized Shiga toxin effectorpolypeptide derived from the A subunit of Shiga-like Toxin 1 (SLT-1A).An immunoglobulin-type binding region αUL18 is derived from an antibodygenerated, using techniques known in the art, to the cell-surfacecytomegalovirus protein UL18, which is present on human cells infectedwith cytomegalovirus (Yang Z, Bjorkman P, Proc Natl Acad Sci USA 105:10095-100 (2008)). The human cytomegalovirus infection is associatedwith various cancers and inflammatory disorders.

Construction, Production, and Purification of the Cytotoxic ProteinSLT-1A::αUL18::KDEL

The immunoglobulin-type binding region αUL18 and Shiga toxin effectorregion are linked together, and a carboxy-terminal KDEL is added to forma protein. For example, a fusion protein is produced by expressing apolynucleotide encoding the αUL18-binding protein SLT-1A::αUL18::KDEL.Expression of the SLT-1A::αUL18::KDEL cytotoxic protein is accomplishedusing either bacterial and/or cell-free, protein translation systems asdescribed in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic ProteinSLT-1A::αUL18::KDEL

The binding characteristics of the cytotoxic protein of this example forcytomegalovirus protein UL18 positive cells and cytomegalovirus proteinUL18 negative cells is determined by fluorescence-based, flow-cytometry.The B_(max) for SLT-1A::αUL18::KDEL to cytomegalovirus protein UL18positive cells is measured to be approximately 50,000-200,000 MFI with aK_(D) within the range of 0.01-100 nM, whereas there is no significantbinding to cytomegalovirus protein UL18 negative cells in this assay.

The ribosome inactivation abilities of the SLT-1A::αUL18::KDEL cytotoxicprotein is determined in a cell-free, in vitro protein translation asdescribed above in the previous examples. The inhibitory effect of thecytotoxic protein of this example on cell-free protein synthesis issignificant. The IC₅₀ of SLT-1A::αUL18::KDEL on protein synthesis inthis cell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic ProteinSLT-1A::αUL18::KDEL Using a Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A::αUL18::KDEL are determinedby the general cell-kill assay as described above in the previousexamples using cytomegalovirus protein UL18 positive cells. In addition,the selective cytotoxicity characteristics of SLT-1A::αUL18::KDEL aredetermined by the same general cell-kill assay using cytomegalovirusprotein UL18 negative cells as a comparison to the cytomegalovirusprotein UL18 positive cells. The CD₅₀ of the cytotoxic protein of thisexample is approximately 0.01-100 nM for cytomegalovirus protein UL18positive cells depending on the cell line. The CD₅₀ of the cytotoxicprotein is approximately 10-10,000 fold greater (less cytotoxic) forcells not expressing the cytomegalovirus protein UL18 on a cellularsurface as compared to cells which do express the cytomegalovirusprotein UL18 on a cellular surface. In addition, the cytotoxicity ofSLT-1A::αUL18::KDEL is investigated for both direct cytotoxicity andindirect cytotoxicity by T-cell epitope delivery and presentationleading to CTL-mediated cytotoxicity.

Example 15 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Diphtheria Toxin Effector Polypeptideand a Binding Region Specific to CD20 (αCD20 Fused with DiphtheriaToxin)

In this example, a CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cellde-immunized diphtheria toxin effector region is derived from the Asubunit of diphtheria toxin 1 as described above. An immunoglobulin-typebinding region αCD20-antigen is derived from an immunoglobulin-typedomain recognizing human CD20 (see e.g. Haisma et al., Blood 92: 184-90(1999); Geng S et al., Cell Mol Immunol 3: 439-43 (2006); Olafesn T etal., Protein Eng Des Sel 23: 243-9 (2010)), which comprises animmunoglobulin-type binding region capable of binding an extracellularpart of CD20. CD20 is expressed on multiple cancer cell types, such as,e.g., B-cell lymphoma cells, hairy cell leukemia cells, B-cell chroniclymphocytic leukemia cells, and melanoma cells. In addition, CD20 is anattractive target for therapeutics to treat certain autoimmune diseases,disorders, and conditions involving overactive B-cells.

Construction, Production, and Purification of the Cytotoxic ProteinDiphtheria Toxin::αCD20

The immunoglobulin-type binding region αCD20 and diphtheria toxineffector region (such as, e.g., SEQ ID NOs: 46, 47, and 48) are linkedtogether. For example, a fusion protein is produced by expressing apolynucleotide encoding the αCD20-antigen-binding protein diphtheriatoxin::αCD20 (see, e.g., SEQ ID NOs: 55, 56, and 57). Expression of theSLT diphtheria toxin::αCD20 cytotoxic protein is accomplished usingeither bacterial and/or cell-free, protein translation systems asdescribed in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic ProteinDiphtheria Toxin::αCD20

The binding characteristics of the cytotoxic protein of this example forCD20+ cells and CD20− cells is determined by fluorescence-based,flow-cytometry assay as described in previous patents. The B_(max) fordiphtheria toxin::αCD20 to CD20+ cells is measured to be approximately50,000-200,000 MFI with a K_(D) within the range of 0.01-100 nM, whereasthere is no significant binding to CD20− cells in this assay.

The ribosome inactivation abilities of the diphtheria toxin::αCD20cytotoxic protein is determined in a cell-free, in vitro proteintranslation as described above in the previous examples. The inhibitoryeffect of the cytotoxic protein of this example on cell-free proteinsynthesis is significant. The IC₅₀ of diphtheria toxin::αCD20 on proteinsynthesis in this cell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic Protein DiphtheriaToxin::αCD20 Using a CD20+ Cell-Kill Assay

The cytotoxicity characteristics of diphtheria toxin::αCD20 aredetermined by the general cell-kill assay as described above in theprevious examples using CD20+ cells. In addition, the selectivecytotoxicity characteristics of diphtheria toxin::αCD20 are determinedby the same general cell-kill assay using CD20− cells as a comparison tothe CD20+ cells. The CD₅₀ of the cytotoxic protein of this example isapproximately 0.01-100 nM for CD20+ cells depending on the cell line.The CD₅₀ of the cytotoxic protein is approximately 10-10,000 foldgreater (less cytotoxic) for cells not expressing CD20 on a cellularsurface as compared to cells which do express CD20 on a cellularsurface. In addition, the cytotoxicity of diphtheria toxin::αCD20 isinvestigated for both direct cytotoxicity and indirect cytotoxicity byT-cell epitope delivery and presentation leading to CTL-mediatedcytotoxicity.

Determining the In Vivo Effects of the Cytotoxic Protein DiphtheriaToxin::αCD20 Using Animal Models

Animal models are used to determine the in vivo effects of the cytotoxicprotein diphtheria toxin::αCD20 on neoplastic cells. Various micestrains are used to test the effect of the cytotoxic protein afterintravenous administration on xenograft tumors in mice resulting fromthe injection into those mice of human neoplastic cells which expressCD20 on their cell surfaces. Cell killing is investigated for bothdirect cytotoxicity and indirect cytotoxicity by T-cell epitope deliveryand presentation leading to CTL-mediated cytotoxicity.

Example 16 A Cytotoxic Protein Comprising a T-Cell Hyper-Immunized andB-Cell/CD4+ T-Cell De-Immunized Diphtheria Toxin Effector Polypeptideand a Binding Region Specific to HER2 (“αHER2-V_(H)H Fused withDiphtheria Toxin”)

In this example, the CD8+ T-cell hyper-immunized and B-cell/CD4+ T-cellde-immunized diphtheria toxin effector region is derived from the Asubunit of diphtheria toxin as described above. The immunoglobulin-typebinding region is αHER2 V_(H)H derived from a single-domain variableregion of the camelid antibody (V_(H)H) protein 5F7, as described inU.S. Patent Application Publication 2011/0059090.

Construction, Production, and Purification of the Cytotoxic Protein“αHER2-V_(H)H Fused with Diphtheria Toxin”

The immunoglobulin-type binding region and diphtheria toxin effectorregion are linked together to form a fused protein (see, e.g., SEQ IDNOs: 58, 59, and 60). In this example, a polynucleotide encoding theαHER2-V_(H)H variable region derived from protein 5F7 may be cloned inframe with a polynucleotide encoding a linker known in the art and inframe with a polynucleotide encoding the diphtheria toxin effectorregion comprising amino acids of SEQ ID NOs: 46, 47, or 48. Variants of“αHER2-V_(H)H fused with diphtheria toxin” cytotoxic proteins arecreated such that the binding region is optionally located adjacent tothe amino-terminus of the diphtheria toxin effector region andoptionally comprises a carboxy-terminal endoplasmic reticulum signalmotif of the KDEL family. Expression of the “αHER2-V_(H)H fused withdiphtheria toxin” cytotoxic protein variants is accomplished usingeither bacterial and/or cell-free, protein translation systems asdescribed in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic Proteins“αHER2-V_(H)H Fused with Diphtheria Toxin”

The binding characteristics of the cytotoxic protein of this example forHER2+ cells and HER2− cells is determined by fluorescence-based,flow-cytometry assay as described in previous patents. The B_(max) for“αHER2-V_(H)H fused with diphtheria toxin” to HER2+ cells is measured tobe approximately 50,000-200,000 MFI with a K_(D) within the range of0.01-100 nM, whereas there is no significant binding to HER2− cells inthis assay.

The ribosome inactivation abilities of the “αHER2-V_(H)H fused withdiphtheria toxin” cytotoxic proteins is determined in a cell-free, invitro protein translation as described above in the previous examples.The inhibitory effect of the cytotoxic protein of this example oncell-free protein synthesis is significant. The IC₅₀ of “αHER2-V_(H)Hfused with diphtheria toxin” on protein synthesis in this cell-freeassay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cytotoxic Protein “αHER2-V_(H)HFused with Diphtheria Toxin” Using a HER2+ Cell-Kill Assay

The cytotoxicity characteristics of “αHER2-V_(H)H fused with diphtheriatoxin” are determined by the general cell-kill assay as described abovein the previous examples using HER2+ cells. In addition, the selectivecytotoxicity characteristics of “αHER2-V_(H)H fused with diphtheriatoxin” are determined by the same general cell-kill assay using HER2−cells as a comparison to the HER2+ cells. The CD₅₀ of the cytotoxicprotein of this example is approximately 0.01-100 nM for HER2+ cellsdepending on the cell line. The CD₅₀ of the cytotoxic protein isapproximately 10-10,000 fold greater (less cytotoxic) for cells notexpressing HER2 on a cellular surface as compared to cells which doexpress HER2 on a cellular surface. In addition, the cytotoxicity of“αHER2-V_(H)H fused with diphtheria toxin” is investigated for bothdirect cytotoxicity and indirect cytotoxicity by T-cell epitope deliveryand presentation leading to CTL-mediated cytotoxicity.

Determining the In Vivo Effects of the Cytotoxic Protein “αHER2-V_(H)HFused with Diphtheria Toxin” Using Animal Models

Animal models are used to determine the in vivo effects of the cytotoxicprotein “αHER2-V_(H)H fused with diphtheria toxin” on neoplastic cells.Various mice strains are used to test the effect of the cytotoxicprotein after intravenous administration on xenograft tumors in miceresulting from the injection into those mice of human neoplastic cellswhich express HER2 on their cell surfaces. Cell killing is investigatedfor both direct cytotoxicity and indirect cytotoxicity by T-cell epitopedelivery and presentation leading to CTL-mediated cytotoxicity.

Example 17 T-Cell Hyper-Immunized and/or B-Cell/CD4+ T-Cell De-ImmunizedShiga Toxin Derived Cytotoxic Proteins Targeting Various Cell Types

In this example, the Shiga toxin effector region comprises T-cellhyper-immunized and/or B-cell/CD4+ T-cell de-immunized Shiga toxineffector polypeptide derived from the A subunit of Shiga-like Toxin 1(SLT-1A), Shiga toxin (StxA), and/or Shiga-like Toxin 2 (SLT-2A) withany one or more of the aforementioned B-cell epitope regions disruptedvia one or more embedded or inserting T-cell epitopes. A binding regionis derived from the immunoglobulin domain from the molecule chosen fromcolumn 1 of Table 15 and which binds the extracellular targetbiomolecule indicated in column 2 of Table 15. The exemplary cytotoxicproteins of this example are optionally created with a carboxy-terminalKDEL-type signal motif and/or detection promoting agent(s) usingreagents and techniques known in the art. The exemplary cytotoxicproteins of this example are tested as described in the previousexamples using cells expressing the appropriate extracellular targetbiomolecules. The exemplary proteins of this example may be used, e.g.,to diagnose and treat diseases, conditions, and/or disorders indicatedin column 3 of Table 15.

TABLE 15 Various Binding Regions for Cell Targeting of CytotoxicProteins Source of binding region Extracellular target Application(s)alemtuzumab CD52 B-cell cancers, such as lymphoma and leukemia, andB-cell related immune disorders, such as autoimmune disordersbasiliximab CD25 T-cell disorders, such as prevention of organtransplant rejections, and some B-cell lineage cancers brentuximab CD30hematological cancers, B-cell related immune disorders, and T-cellrelated immune disorders catumaxomab EpCAM various cancers, such asovarian cancer, malignant ascites, gastric cancer cetuximab EGFR variouscancers, such as colorectal cancer and head and neck cancer daclizumabCD25 B-cell lineage cancers and T-cell disorders, such as rejection oforgan transplants daratumumab CD38 hematological cancers, B-cell relatedimmune disorders, and T-cell related immune disorders dinutuximabganglioside GD2 Various cancers, such as breast cancer, myeloid cancers,and neuroblastoma efalizumab LFA-1 (CD11a) autoimmune disorders, such aspsoriasis ertumaxomab HER2/neu various cancers and tumors, such asbreast cancer and colorectal cancer gemtuzumab CD33 myeloid cancer orimmune disorder ibritumomab CD20 B-cell cancers, such as lymphoma andleukemia, and B-cell related immune disorders, such as autoimmunedisorders ipilimumab CD152 T-cell related disorders and various cancers,such as leukemia, melanoma muromonab CD3 prevention of organ transplantrejections natalizumab integrin α4 autoimmune disorders, such asmultiple sclerosis and Crohn's disease obinutuzumab CD20 B-cell cancers,such as lymphoma and leukemia, and B-cell related immune disorders, suchas autoimmune disorders ocaratuzumab CD20 B-cell cancers, such aslymphoma and leukemia, and B-cell related immune disorders, such asautoimmune disorders ocrelizumab CD20 B-cell cancers, such as lymphomaand leukemia, and B-cell related immune disorders, such as autoimmunedisorders ofatumumab CD20 B-cell cancers, such as lymphoma and leukemia,and B-cell related immune disorders, such as autoimmune disorderspalivizumab F protein of respiratory treat respiratory syncytial virussyncytial virus panitumumab EGFR various cancers, such as colorectalcancer and head and neck cancer pertuzumab HER2/neu various cancers andtumors, such as breast cancer and colorectal cancer pro 140 CCR5 HIVinfection and T-cell disorders ramucirumab VEGFR2 various cancers andcancer related disorders, such as solid tumors rituximab CD20 B-cellcancers, such as lymphoma and leukemia, and B-cell related immunedisorders, such as autoimmune disorders tocilizumab or IL-6 receptorautoimmune disorders, atlizumab such as rheumatoid arthritis tositumomabCD20 B-cell cancers, such as lymphoma and leukemia, and B-cell relatedimmune disorders, such as autoimmune disorders trastuzumab HER2/neuvarious cancers and tumors, such as breast cancer and colorectal cancerublituximab CD20 B-cell cancers, such as lymphoma and leukemia, andB-cell related immune disorders, such as autoimmune disordersvedolizumab integrin α4β7 autoimmune disorders, such as Crohn's diseaseand ulcerative colitis CD20 binding scFv(s) CD20 B-cell cancers, such asGeng S et al., Cell Mol lymphoma and Immunol 3: 439-43 leukemia, andB-cell (2006); Olafesn T et related immune al., Protein Eng Desdisorders, such as Sel 23: 243-9 (2010) autoimmune disorders CD22binding scFv(s) CD22 B-cell cancers or B-cell Kawas S et al., MAbsrelated immune 3: 479-86 (2011) disorders CD25 binding scFv(s) CD25various cancers of the Muramatsu H et al., B-cell lineage and CancerLett 225: 225- immune disorders 36 (2005) related to T-cells CD30binding CD30 B-cell cancers or B- monoclonal cell/T-cell relatedantibody(s) immune disorders Klimka A et al., Br J Cancer 83: 252-60(2000) CD33 binding CD33 myeloid cancer or monoclonal immune disorderantibody(s) Benedict C et al., J Immunol Methods 201: 223-31 (1997) CD38binding CD38 hematological cancers, immunoglobulin B-cell related immunedomains U.S. Pat No. disorders, and T-cell 8,153,765 related immunedisorders CD40 binding scFv(s) CD40 various cancers and Ellmark P etal., immune disorders Immunology 106: 456- 63 (2002) CD52 binding CD52B-cell cancers, such as monoclonal lymphoma and antibody(s) leukemia,and B-cell U.S. Pat. No. 7,910,104 related immune B2 disorders, such asautoimmune disorders CD56 binding CD56 immune disorders and monoclonalvarious cancers, such as antibody(s) lung cancer, Merkel cell Shin J etal., carcinoma, myeloma Hybridoma 18: 521-7 (1999) CD79 binding CD79B-cell cancers or B-cell monoclonal related immune antibody(s) disordersZhang L et al., Ther Immunol 2: 191-202 (1995) CD248 binding CD248various cancers, such as scFv(s) inhibiting angiogenesis Zhao A et al.,J Immunol Methods 363: 221-32 (2011) EpCAM binding EpCAM variouscancers, such as monoclonal ovarian cancer, antibody(s) malignantascites, Schanzer J et al., J gastric cancer Immunother 29: 477- 88(2006) PSMA binding PSMA prostate cancer monoclonal antibody(s) FrigerioB et al., Eur J Cancer 49: 2223-32 (2013) Eph-B2 binding Eph-B2 forvarious cancers such monoclonal as colorectal cancer and antibody(s)prostate cancer Abéngozar M et al., Blood 119: 4565-76 (2012) Endoglinbinding Endoglin various cancers, such as monoclonal breast cancer andantibody(s) colorectal cancers Völkel T et al., Biochim Biophys Res Acta1663: 158-66 (2004) FAP binding FAP various cancers, such as monoclonalsarcomas and bone antibody(s) cancers Zhang J et al., FASEB J 27: 581-9(2013) CEA binding CEA various cancers, such as antibody(s) andgastrointestinal cancer, scFv(s) pancreatic cancer, lung Neumaier M etal., cancer, and breast Cancer Res 50: 2128- cancer 34 (1990); Pavoni Eet al., BMC Cancer 6: 4 (2006); Yazaki P et al., Nucl Med Biol 35: 151-8(2008); Zhao J et al., Oncol Res 17: 217-22 (2008) CD24 binding CD24various cancers, such as monoclonal bladder cancer antibody(s)Kristiansen G et al., Lab Invest 90: 1102-16 (2010) LewisY antigenLewisY antigens various cancers, such as binding scFv(s) cervical cancerand Power B et al., Protein uterine cancer Sci 12: 734-47 (2003);monoclonal antibody BR96 Feridani A et al., Cytometry 71: 361-70 (2007)adalimumab TNF-α various cancers and immune disorders, such asRheumatoid arthritis, Crohn's Disease, Plaque Psoriasis, PsoriaticArthritis, Ankylosing Spondylitis, Juvenile Idiopathic Arthritis,Hemolytic disease of the newborn afelimomab TNF-α various cancers andimmune disorders ald518 IL-6 various cancers and immune disorders, suchas rheumatoid arthritis anrukinzumab or ima- IL-13 various cancers and638 immune disorders briakinumab IL-12, IL-23 various cancers and immunedisorders, such as psoriasis, rheumatoid arthritis, inflammatory boweldiseases, multiple sclerosis brodalumab IL-17 various cancers and immunedisorders, such as inflammatory diseases canakinumab IL-1 variouscancers and immune disorders, such as rheumatoid arthritis certolizumabTNF-α various cancers and immune disorders, such as Crohn's diseasefezakinumab IL-22 various cancers and immune disorders, such asrheumatoid arthritis, psoriasis ganitumab IGF-I various cancersgolimumab TNF-α various cancers and immune disorders, such as rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis infliximab TNF-αvarious cancers and immune disorders, such as rheumatoid arthritis,ankylosing spondylitis, psoriatic arthritis, psoriasis, Crohnαs disease,ulcerative colitis ixekizumab IL-17A various cancers and immunedisorders, such as autoimmune diseases mepolizumab IL-5 various immunedisorders and cancers, such as B-cell cancers nerelimomab TNF-α variouscancers and immune disorders olokizumab IL6 various cancers and immunedisorders ozoralizumab TNF-α inflammation perakizumab IL17A variouscancers and immune disorders, such as arthritis placulumab human TNFvarious immune disorders and cancers sarilumab IL6 various cancers andimmune disorders, such as rheumatoid arthritis, ankylosing spondylitissiltuximab IL-6 various cancers and immune disorders sirukumab IL-6various cancers and immune disorders, such as rheumatoid arthritistabalumab BAFF B-cell cancers ticilimumab or CTLA-4 various cancerstremelimumab tildrakizumab IL23 immunologically mediated inflammatorydisorders tnx-650 IL-13 various cancers and immune disorders, such asB-cell cancers tocilizumab or IL-6 receptor various cancers andatlizumab immune disorders, such as rheumatoid arthritis ustekinumabIL-12, IL-23 various cancers and immune disorders, such as multiplesclerosis, psoriasis, psoriatic arthritis Various growth VEGFR, EGFR,various cancer, such as factors: VEGF, EGF1, FGFR breast cancer andcolon EGF2, FGF cancer, and to inhibit vascularization Variouscytokines: IL- IL-2R, IL-6R, IL-23R, various immune 2, IL-6, IL-23,CCL2, CD80/CD86, disorders and cancers BAFFs, TNFs, TNFRSF13/ RANKLTNFRSF17, TNFR Broadly neutralizing Influenza surface viral infectionsantibodies identified antigens, e.g. from patient samples Prabakaran etal., hemaglutinins and Front Microbiol 3: 277 influenza matrix (2012)protein 2 Broadly neutralizing Coronavirus surface viral infectionsantibodies identified antigens from patient samples Prabakaran et al.,Front Microbiol 3: 277 (2012) Broadly neutralizing Henipaviruses surfaceviral infections antibodies identified antigens from patient samplesPrabakaran et al., Front Microbiol 3: 277 (2012)

While some embodiments of the invention have been described by way ofillustration, it will be apparent that the invention can be put intopractice with many modifications, variations and adaptations, and withthe use of numerous equivalents or alternative solutions that are withinthe scope of persons skilled in the art, without departing from thespirit of the invention or exceeding the scope of the claims.

All publications, patents, and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. The disclosures of U.S. provisional patent applicationSer. Nos. 61/777,130, 61/932,000, 61/951,110, 61/951,121, 62/010,918,and 62/049,325 are each incorporated herein by reference in theirentirety. The disclosures of U.S. patent application publications US2007/0298434 A1, US 2009/0156417 A1, and US 2013/0196928 A1 are eachincorporated here by reference in their entirety. The disclosures ofinternational PCT patent application serial numbers PCT/US2014/023231and PCT/US2014/023198 are each incorporated herein by reference in theirentirety. The complete disclosures of all electronically availablebiological sequence information from GenBank (National Center forBiotechnology Information, U.S.) for amino acid and nucleotide sequencescited herein are each incorporated herein by reference in theirentirety.

The invention is claimed as follows: 1-71. (canceled)
 106. A polypeptidecomprising an embedded, heterologous, CD8+ T-cell epitope, wherein thepolypeptide is capable of intracellular delivery of the T-cell epitopefrom an early endosomal compartment to a MHC class I molecule of a cellin which the polypeptide is present; and wherein the embedded,heterologous CD8+ T-cell epitope replaces an equivalent number of aminoacid residues in a parental polypeptide such that the polypeptidecomprising the epitope has the same total number of amino acids as theparental polypeptide.
 107. The polypeptide of claim 106, comprising aproteasome delivering effector polypeptide.
 108. The polypeptide ofclaim 106, comprising a toxin effector polypeptide capable of exhibitingone or more toxin effector functions.
 109. The polypeptide of claim 108,wherein the toxin effector polypeptide comprises a proteasome deliveringeffector polypeptide.
 110. The polypeptide of claim 108 or claim 109,wherein the heterologous, CD8+ T-cell epitope is embedded in the toxineffector polypeptide.
 111. The polypeptide of claim 110, wherein thetoxin effector polypeptide is capable of exhibiting one or more toxineffector functions in addition to intracellular delivery of a CD8+T-cell epitope from an early endosomal compartment to a MHC class Imolecule of a cell in which the toxin effector polypeptide is present.112. The polypeptide of any one of claims 108-111, wherein the toxineffector polypeptide is derived from a toxin selected from the groupconsisting of: ABx toxin, ribosome inactivating protein toxin, abrin,anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin,diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin,pertussis toxin, pokeweed antiviral protein, pulchellin, Pseudomonasexotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, andsubtilase cytotoxin.
 113. The polypeptide of claim 112, wherein thetoxin effector polypeptide is derived from a polypeptide selected fromthe group of polypeptides represented by: (i) amino acids 75 to 251 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; (ii) amino acids 2 to 389 ofSEQ ID NO:45; (iii) amino acids 1 to 241 of SEQ ID NO: 1, SEQ ID NO:2,or SEQ ID NO:3; (iv) amino acids 1 to 251 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; and (v) amino acids 1 to 261 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO:3.
 114. A polypeptide comprising an embedded,heterologous, CD8+ T-cell epitope disrupting an endogenous B-cellepitope and/or CD4+ T-cell epitope, wherein the embedded, heterologousCD8+ T-cell epitope replaces an equivalent number of amino acid residuesin a parental polypeptide such that the polypeptide comprising theepitope has the same total number of amino acids as the parentalpolypeptide.
 115. The polypeptide of claim 114, wherein the polypeptideis capable of intracellular delivery of the CD8+ T-cell epitope to a MHCclass I molecule from an early endosomal compartment of a cell in whichthe polypeptide is present.
 116. The polypeptide of claim 114 or claim115, comprising a toxin effector polypeptide capable of exhibiting oneor more toxin effector functions.
 117. The polypeptide of claim 116,wherein the heterologous, CD8+ T-cell epitope is embedded in the toxineffector polypeptide.
 118. The polypeptide of claim 117, wherein thetoxin effector polypeptide is capable of exhibiting one or more toxineffector functions in addition to intracellular delivery of a CD8+T-cell epitope from an early endosomal compartment to a MHC class Imolecule of a cell in which the toxin effector polypeptide is present.119. The polypeptide of claim 116, wherein the toxin effectorpolypeptide is derived from a toxin selected from the group consistingof: ABx toxin, ribosome inactivating protein toxin, abrin, anthraxtoxin, Aspfl, bouganin, bryodin, cholix toxin, claudin, diphtheriatoxin, gelonin, heat-labile enterotoxin, mitogillin, pertussis toxin,pokeweed antiviral protein, pulchellin, Pseudomonas exotoxin A,restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilasecytotoxin.
 120. The polypeptide of claim 119, wherein the toxin effectorpolypeptide comprises a diphtheria toxin effector polypeptide comprisingamino acid sequences derived from the A and B Subunits of at least onemember of the diphtheria toxin family, wherein the diphtheria toxineffector polypeptide comprises a disruption of at least one B-cellepitope and/or CD4+ T-cell epitope region of the amino acid sequenceselected from the group of natively positioned amino acids consistingof: 3-10 of SEQ ID NO:44, 15-31 of SEQ ID NO:44, 32-54 of SEQ ID NO:44;33-43 of SEQ ID NO:44, 71-77 of SEQ ID NO:44, 93-113 of SEQ ID NO:44,125-131 of SEQ ID NO:44, 138-146 of SEQ ID NO:44, 141-167 of SEQ IDNO:44, 165-175 of SEQ ID NO:44, 182-201 of SEQ ID NO:45, 185-191 of SEQID NO:44, and 225-238 of SEQ ID NO:45; and wherein the diphtheria toxineffector polypeptide is capable of routing to a cytosol compartment of acell in which the diphtheria toxin effector polypeptide is present. 121.The polypeptide of claim 119, wherein the diphtheria toxin effectorpolypeptide is derived from the polypeptide represented by amino acids 2to 389 of SEQ ID NO:45.
 122. The polypeptide of claim 119, wherein thetoxin effector polypeptide comprises a Shiga toxin effector polypeptidecomprising an amino acid sequence derived from an A Subunit of at leastone member of the Shiga toxin family, wherein the Shiga toxin effectorpolypeptide comprises a disruption of at least one B-cell epitope and/orCD4+ T-cell epitope region of the Shiga toxin A Subunit amino acidsequence selected from the group of natively positioned amino acidsconsisting of: the B-cell epitope regions: 1-15 of SEQ ID NO: 1 or SEQID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ IDNO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ IDNO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ IDNO:2; and 210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQID NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1 orSEQ ID NO:2, and the CD4+ T-cell epitope regions: 4-33 of SEQ ID NO: 1or SEQ ID NO:2, 34-78 of SEQ ID NO: 1 or SEQ ID NO:2, 77-103 of SEQ IDNO: 1 or SEQ ID NO:2, 128-168 of SEQ ID NO: 1 or SEQ ID NO:2, 160-183 ofSEQ ID NO:1 or SEQ ID NO:2, 236-258 of SEQ ID NO: 1 or SEQ ID NO:2, and274-293 of SEQ ID NO: 1 or SEQ ID NO:2; and wherein the Shiga toxineffector polypeptide is capable of routing to a cytosol compartment of acell in which the Shiga toxin effector polypeptide is present.
 123. Thepolypeptide of claim 119, wherein the Shiga toxin effector polypeptideis derived from a polypeptide selected from the group of polypeptidesrepresented by: (i) amino acids 75 to 251 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; (ii) amino acids 1 to 241 of SEQ ID NO: 1, SEQ ID NO:2,or SEQ ID NO:3; (iii) amino acids 1 to 251 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; and (iv) amino acids 1 to 261 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3.
 124. A method of creating a CD8+ T-cell epitopedelivery molecule capable of intracellular delivery of a T-cell epitopefrom an early endosomal compartment to a MHC class I molecule of a cellin which the delivery molecule is present, the method comprising thestep of: embedding a heterologous, CD8+ T-cell epitope in a proteasomedelivering effector polypeptide capable of intracellular delivery of aT-cell epitope from an early endosomal compartment to a MHC class Imolecule of a cell in which the delivery molecule is present; whereinthe step of embedding involves replacing an equivalent number of aminoacid residues in a parental polypeptide with the heterologous, CD8+T-cell epitope such that the polypeptide comprising the epitope has thesame total number of amino acids as the parental polypeptide.
 125. Themethod of claim 124, wherein the method comprises embedding the CD8+T-cell epitope in an endogenous B-cell epitope, an endogenous CD4+T-cell epitope, and/or a catalytic domain of the proteasome deliveringeffector polypeptide.
 126. The method of claim 124 or claim 125, whereinthe CD8+ T-cell epitope delivery molecule comprises a toxin effectorpolypeptide comprising a proteasome delivering effector polypeptide.127. The method of claim 126, wherein the method comprises embedding theheterologous, CD8+ T-cell epitope in the toxin effector polypeptide.128. The method of claim 127, wherein the embedding step results in aCD8+ T-cell epitope delivery molecule comprising a toxin effectorpolypeptide capable of exhibiting one or more toxin effector functionsin addition to intracellular delivery of a CD8+ T-cell epitope from anearly endosomal compartment to a MHC class I molecule of a cell in whichthe toxin effector polypeptide is present.
 129. A method for reducingB-cell immunogenicity of a polypeptide having a B-cell epitope, themethod comprising the step of: disrupting the B-cell epitope in thepolypeptide with one or more amino acid residue(s) of a T-cell epitopeembedded in the polypeptide; wherein the T-cell epitope is embedded inthe polypeptide by replacing an equivalent number of amino acid residuesin a parental polypeptide with the T-cell epitope such that thepolypeptide comprising the epitope has the same total number of aminoacids as the parental polypeptide.
 130. A method for reducing B-cellimmunogenicity of a polypeptide having a B-cell epitope whilesimultaneously increasing CD8+ T-cell immunogenicity of the polypeptide,the method comprising the step of: disrupting a B-cell epitope in thepolypeptide with one or more amino acid residue(s) of a heterologous,CD8+ T-cell epitope embedded in the polypeptide; wherein the CD8+ T-cellepitope is embedded in the polypeptide by replacing an equivalent numberof amino acid residues in a parental polypeptide with the heterologous,CD8+ T-cell epitope such that the polypeptide comprising the epitope hasthe same total number of amino acids as the parental polypeptide. 131.The method of claim 129 or claim 130, wherein the polypeptide has a CD4+T-cell epitope, and wherein the B-cell epitope disrupting step comprisesmaking one or more amino acid substitutions in the CD4+ T-cell epitope.132. A method for reducing CD4+ T-cell immunogenicity of a polypeptidehaving a CD4+ T-cell epitope, the method comprising the step of:disrupting a CD4+ T-cell epitope in the polypeptide with one or moreamino acid residue(s) of a CD8+ T-cell epitope embedded in thepolypeptide; wherein the CD8+ T-cell epitope is embedded in thepolypeptide by replacing an equivalent number of amino acid residues ina parental polypeptide with the CD8+ T-cell epitope such that thepolypeptide comprising the epitope has the same total number of aminoacids as the parental polypeptide.
 133. A method for reducing CD4+T-cell immunogenicity of a polypeptide having a CD4+ T-cell epitopewhile simultaneously increasing CD8+ T-cell immunogenicity of thepolypeptide, the method comprising the step of: disrupting a CD4+ T-cellepitope with one or more amino acid residue(s) of a heterologous, CD8+T-cell epitope embedded in the polypeptide; wherein the CD8+ T-cellepitope is embedded in the polypeptide by replacing an equivalent numberof amino acid residues in a parental polypeptide with the heterologous,CD8+ T-cell epitope such that the polypeptide comprising the epitope hasthe same total number of amino acids as the parental polypeptide. 134.The method of claim 132 or claim 133, wherein the polypeptide has aB-cell epitope, and wherein the CD4+ T-cell epitope disrupting stepcomprises making one or more amino acid substitutions in the B-cellepitope.
 135. A polypeptide comprising or consisting essentially of thepolypeptide shown in any one of SEQ ID NOs: 11-13, 15-19, 21-43, or46-48.